216 research outputs found
Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated
Background: Retrograde transport of several transmembrane proteins from endosomes to the trans-Golgi network (TGN) occurs via Rab 5-containing endosomes, mediated by clathrin and the recently characterized retromer complex. This complex and one of its putative sorting receptor components, SorLA, were reported to be associated to late onset Alzheimer's disease (AD). The pathogenesis of this neurodegenerative disorder is still elusive, although accumulation of amyloidogenic Abeta is a hallmark. This peptide is generated from the sucessive β- and γ- secretase proteolysis of the Alzheimer's amyloid precursor protein (APP), events which are associated with endocytic pathway compartments. Therefore, APP targeting and time of residence in endosomes would be predicted to modulate Abeta levels. However, the formation of an APP- and retromer-containing protein complex with potential functions in retrieval of APP from the endosome to the TGN had, to date, not been demonstrated directly. Further, the motif(s) in APP that regulate its sorting to the TGN have not been characterized. Results: Through the use of APP-GFP constructs, we show that APP containing endocytic vesicles targeted for the TGN, are also immunoreactive for clathrin-, Rab 5- and VPS35. Further, they frequently generate protruding tubules near the TGN, supporting an association with a retromer-mediated pathway. Importantly, we show for the first time, that mimicking APP phosphorylation at S655, within the APP 653YTSI656 basolateral motif, enhances APP retrieval via a retromer-mediated process. The phosphomimetic APP S655E displays decreased APP lysosomal targeting, enhanced mature half-life, and decreased tendency towards Abeta production. VPS35 downregulation impairs the phosphorylation dependent APP retrieval to the TGN, and decreases APP half-life. Conclusions: We reported for the first time the importance of APP phosphorylation on S655 in regulating its retromer-mediated sorting to the TGN or lysosomes. Significantly, the data are consistent with known interactions involving the retromer, SorLA and APP. Further, these findings add to our understanding of APP targeting and potentially contribute to our knowledge of sporadic AD pathogenesis representing putative new targets for AD therapeutic strategies
Impact of mesenchymal stem cells' secretome on glioblastoma pathophysiology
Background: Glioblastoma (GBM) is a highly aggressive primary brain cancer, for which curative therapies are not available. An emerging therapeutic approach suggested to have potential to target malignant gliomas has been based on the use of multipotent mesenchymal stem cells (MSCs), either unmodified or engineered to deliver anticancer therapeutic agents, as these cells present an intrinsic capacity to migrate towards malignant tumors. Nevertheless, it is still controversial whether this innate tropism of MSCs towards the tumor area is associated with cancer promotion or suppression. Considering that one of the major mechanisms by which MSCs interact with and modulate tumor cells is via secreted factors, we studied how the secretome of MSCs modulates critical hallmark features of GBM cells.
Methods: The effect of conditioned media (CM) from human umbilical cord perivascular cells (HUCPVCs, a MSC population present in the Wharton's jelly of the umbilical cord) on GBM cell viability, migration, proliferation and sensitivity to temozolomide treatment of U251 and SNB-19 GBM cells was evaluated. The in vivo chicken chorioallantoic membrane (CAM) assay was used to evaluate the effect of HUCPVCs CM on tumor growth and angiogenesis. The secretome of HUCPVCs was characterized by proteomic analyses.
Results: We found that both tested GBM cell lines exposed to HUCPVCs CM presented significantly higher cellular viability, proliferation and migration. In contrast, resistance of GBM cells to temozolomide chemotherapy was not significantly affected by HUCPVCs CM. In the in vivo CAM assay, CM from HUCPVCs promoted U251 and SNB-19 tumor cells growth. Proteomic analysis to characterize the secretome of HUCPVCs identified several proteins involved in promotion of cell survival, proliferation and migration, revealing novel putative molecular mediators for the effects observed in GBM cells exposed to HUCPVCs CM.
Conclusions: These findings provide novel insights to better understand the interplay between GBM cells and MSCs, raising awareness to potential safety issues regarding the use of MSCs as stem-cell based therapies for GBM.The authors would like to acknowledge the funding agencies that supported this work: Fundacao para a Ciencia e Tecnologia (FCT), Portugal, projects reference: PTDC/SAU-GMG/113795/2009 (BMC); SFRH/BD/88121/2012 (JVdC); SFRH/BD/103075/2014 (EDG); IF/00601/2012 (BMC); IF/00111/2013 (AJS); SFRH/BD/81495/2011 (SIA); PTDC/NEU-NMC/0205/2012, PTDC/NEUSCC/ 7051/2014, PEst-C/SAU/LA0001/2013-2014 and UID/NEU/04539/2013 (BM); Fundacao Calouste Gulbenkian (BMC); Liga Portuguesa Contra o Cancro (BMC); " COMPETE Programa Operacional Factores de Competitividade, QREN, the European Union (FEDER-Fundo Europeu de Desenvolvimento Regional) and by The National Mass Spectrometry Network (RNEM) under the contract REDE/1506/REM/2005; FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038; and project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). The funding body did not have a role in the design of the study, in collection, analysis or interpretation of data, or in writing the manuscript
Steroid–quinoline hybrids for disruption and reversion of protein aggregation processes
Reversing protein aggregation within cells may be an
important tool to fight protein-misfolding disorders such as Alzheimer’s,
Parkinson’s, and cardiovascular diseases. Here we report the design and
synthesis of a family of steroid−quinoline hybrid compounds based on
the framework combination approach. This set of hybrid compounds
effectively inhibited Aβ1−42 self-aggregation in vitro by delaying the
exponential growth phase and/or reducing the quantity of fibrils in the
steady state. Their disaggregation efficacy was further demonstrated
against preaggregated Aβ1−42 peptides in cellular assays upon their
endocytosis by neuroblastoma cells, as they reverted both the number
and the average area of fibrils back to basal levels. The antiaggregation
effect of these hybrids was further tested and demonstrated in a cellular
model of general protein aggregation expressing a protein aggregation fluorescent sensor. Together, our results show that the new
cholesterol−quinoline hybrids possess wide and marked disaggregation capacities and are therefore promising templates for the
development of new drugs to deal with conformational disorders.Thanks are due to the University of Aveiro, FCT/MEC,
Centro 2020 and Portugal2020, the COMPETE Program, and
the European Union (FEDER Program) via the financial
support to the research units LAQV-REQUIMTE (UIDB/50006/2020), IBiMED (UID/BIM/04501/2019) and CICECO-
Aveiro Institute of Materials (UID/CTM/50011/2019),
financed by national funds through the FCT/MCTES, to the
Portuguese NMR Network, to the ThiMES Project (POCI-01-
0145-FEDER-016630), and to the PAGE Project “Protein
Aggregation Across the Lifespan” (CENTRO-01-0145-
FEDER-000003), including postdoctoral grants to H.M.T.A.
(BPD/UI98/4861/2017) and R.N.d.S. (BPD/UI98/6327/2018). M.P. was supported by Ph.D. Grant SFRH/BD/135655/2018. A.R.S. and S.G. were supported by national
funds (OE) through FCT, I.P., in the scope of the framework
contract foreseen in numbers 4, 5, and 6 of Article 23 of the
Decree-Law 57/2016 of August 29, changed by Law 57/2017
of July 19. Microphotographs were acquired in the LiM facility
of iBiMED/UA, a member of the Portuguese Platform of
BioImaging (PPBI) (POCI-01-0145-FEDER-022122).info:eu-repo/semantics/publishedVersio
Liprobe, a vital dye for lipid aggregates detection in imaging and high-content screens
Pathological lipid accumulation is a hallmark of several metabolic disorders, and
detection of lipid aggregates is an essential step for initial diagnosis and drug
screening purposes. However, low-cost, simple, and reliable detection
fluorescent probes are not widely available. Here, six push-pull-push dyes
were studied, and proved to be highly sensitive to the polarity of the
medium, presenting potential to distinguish structures with different
hydrophobic indexes. Importantly, in the presence of lipid aggregates their
staining specificity highly increased and the fluorescence wavelength blue
shifted. One of the compounds, named Liprobe, was physiologically inert in
cells, as witnessed by mass-spectrometry and metabolic assays. Liprobe was
not toxic to living zebrafish embryos, and differentially stained the muscle and
bone tissues. In triglyceride solutions, a high correlation was observed between
Liprobe’s 558 and 592 nm emissions and the 0–2.5 mg dl−1 triglyceride range.
Confocal and cell-based high content screens revealed that this fluorophore
was able to selectively detect lipid droplets and ceramide loads in normal and
Farber’s disease human fibroblasts, respectively. Our results demonstrate that
Liprobe is a suitable fluorescing probe for vital staining of lipid aggregates,
compatible with a rapid and cheap high content screening assays for
preliminary diagnosis of Farber’s disease and, potentially, of other lipidosis.publishe
CD81 promotes a migratory phenotype in neuronal-like cells
Tetraspanins, such as CD81, can form lateral associations with each other and with other transmembrane proteins. These interactions may underlie CD81 functions in multiple cellular processes, such as adhesion, morphology, migration, and differentiation. Since CD81's role in neuronal cells' migration has not been established, we here evaluated effects of CD81 on the migratory phenotype of SH-SY5Y neuroblastoma cells. CD81 was found enriched at SH-SY5Y cell's membrane, co-localizing with its interactor filamentous-actin (F-actin) in migratory relevant structures of the leading edge (filopodia, stress fibers, and adhesion sites). CD81 overexpression increased the number of cells with a migratory phenotype, in a potentially phosphatidylinositol 3 kinase (PI3K)-Ak strain transforming (AKT) mediated manner. Indeed, CD81 also co-localized with AKT, a CD81-interactor and actin remodeling agent, at the inner leaflet of the plasma membrane. Pharmacologic inhibition of PI3K, the canonical AKT activator, led both to a decrease in the acquisition of a migratory phenotype and to a redistribution of intracellular CD81 and F-actin into cytoplasmic agglomerates. These findings suggest that in neuronal-like cells CD81 bridges active AKT and actin, promoting the actin remodeling that leads to a motile cell morphology. Further studies on this CD81-mediated mechanism will improve our knowledge on important physiological and pathological processes such as cell migration and differentiation, and tumor metastasis.This work was supported by Fundação para a Ciência e
Tecnologia (Portuguese Ministry of Science and Technology), Centro 2020
and Portugal2020, the COMPETE program, QREN, and the European Union
(FEDER program) via the Institute for Biomedicine iBiMED UID/BIM/
04501/2013, fellowship SFRH/BD/90996/2012, project PTDC/CVT-CVT/
32261/2017, and the support of the LiM facility of iBiMED, a member of the
Portuguese Platform of BioImaging (PPBI- POCI-01-0145-FEDER-022122).publishe
TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network
Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood-testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood-testis barrier
Coral symbiotic algae calcify ex hospite in partnership with bacteria
Dinoflagellates of the genus Symbiodinium are commonly recognized
as invertebrate endosymbionts that are of central importance
for the functioning of coral reef ecosystems. However, the
endosymbiotic phase within Symbiodinium life history is inherently
tied to a more cryptic free-living (ex hospite) phase that remains
largely unexplored. Here we show that free-living Symbiodinium
spp. in culture commonly form calcifying bacterial–algal communities
that produce aragonitic spherulites and encase the dinoflagellates
as endolithic cells. This process is driven by Symbiodinium photosynthesis
but occurs only in partnership with bacteria. Our findings
not only place dinoflagellates on the map of microbial–algal
organomineralization processes but also point toward an endolithic
phase in the Symbiodinium life history, a phenomenon that
may provide new perspectives on the biology and ecology of
Symbiodinium spp. and the evolutionary history of the coral–
dinoflagellate symbiosis
Efeito do método de abate e do sexo sobre a qualidade da carne de capivara (Hydrochaeris hydrochaeris)
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