13 research outputs found
Principles of protein targeting to the nucleolus
© 2015 The Author(s). Published with license by Taylor & Francis Group, LLC© Robert M Martin, Gohar Ter-Avetisyan, Henry D Herce, Anne K Ludwig, Gisela Lättig-Tünnemann, and M Cristina Cardoso
This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside the nucleoli of living cells. The data showed that positively charged peptide entities composed of arginines alone and with an isoelectric point at and above 12.6 are necessary and sufficient for mediating significant nucleolar accumulation. A threshold of 6 arginines is necessary for peptides to accumulate in nucleoli, but already 4 arginines are sufficient when fused within 15 amino acid residues of a nuclear localization signal of a protein. Using a pH sensitive dye, we found that the nucleolar compartment is particularly acidic when compared to the surrounding nucleoplasm and, hence, provides the ideal electrochemical environment to bind poly-arginine containing proteins. In fact, we found that oligo-arginine peptides and GFP fusions bind RNA in vitro. Consistent with RNA being the main binding partner for arginines in the nucleolus, we found that the same principles apply to cells from insects to man, indicating that this mechanism is highly conserved throughout evolution.RMM was supported by a post-doctoral grant from Fundação para a Ciência e Tecnologia, Portugal (SFRH-BPD-66611–2009). This work was supported by grants of the German Research Council (DFG CA198/3) to MCC.info:eu-repo/semantics/publishedVersio
Interaction of ARC and Daxx: a novel endogenous target to preserve motor function and cell loss after focal brain ischemia in mice
The aim of this study was to explore the signaling and neuroprotective effect of transactivator of transcription (TAT) protein transduction of the apoptosis repressor with CARD (ARC) in in vitro and in vivo models of cerebral ischemia in mice. In mice, transient focal cerebral ischemia reduced endogenousARCprotein in neurons in the ischemic striatum at early reperfusion time points, and in primary neuronal cultures, RNA interference resulted in greater neuronal susceptibility to oxygen glucose deprivation (OGD).TAT.ARC protein delivery led to a dose-dependent better survival after OGD. Infarct sizes 72 h after 60 min middle cerebral artery occlusion (MCAo) were on average 30±8% (mean±SD; p=0.005; T2-weighted MRI) smaller in TAT.ARC-treated mice (1ug intraventricularly during MCAo) compared with controls. TAT.ARC-treated mice showed better performance in the pole test compared with TAT.β-Gal-treated controls. Importantly, post-stroke treatment (3 h after MCAo) was still effective in affording reduced lesion volume by 20±7% (mean±SD; p˃0.05) and better functional outcome compared with controls. Delayed treatment in mice subjected to 30 min MCAo led to sustained neuroprotection and functional behavior benefits for at least 28 d. Functionally, TAT.ARC treatment inhibited DAXX–ASK1–JNK signaling in the ischemic brain. ARC interacts with DAXX in a CARD-dependent manner to block DAXX trafficking and ASK1–JNK activation. Our work identifies for the first time ARC–DAXX binding to block ASK1–JNK activation as an ARC-specific endogenous mechanism that interferes with neuronal cell death and ischemic brain injury. Delayed delivery of TAT.ARC may present a promising target for stroke therapy
A novel cell permeable DNA replication and repair marker.
Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells
Unique properties of PTEN-L contribute to neuroprotection in response to ischemic-like stress
Abstract Phosphatase and tensin homolog (PTEN) signalling might influence neuronal survival after brain ischemia. However, the influence of the less studied longer variant termed PTEN-L (or PTENα) has not been studied to date. Therefore, we examined the translational variant PTEN-L in the context of neuronal survival. We identified PTEN-L by proteomics in murine neuronal cultures and brain lysates and established a novel model to analyse PTEN or PTEN-L variants independently in vitro while avoiding overexpression. We found that PTEN-L, unlike PTEN, localises predominantly in the cytosol and translocates to the nucleus 10–20 minutes after glutamate stress. Genomic ablation of PTEN and PTEN-L increased neuronal susceptibility to oxygen-glucose deprivation. This effect was rescued by expression of either PTEN-L indicating that both PTEN isoforms might contribute to a neuroprotective response. However, in direct comparison, PTEN-L replaced neurons were protected against ischemic-like stress compared to neurons expressing PTEN. Neurons expressing strictly nuclear PTEN-L NLS showed increased vulnerability, indicating that nuclear PTEN-L alone is not sufficient in protecting against stress. We identified mutually exclusive binding partners of PTEN-L or PTEN in cytosolic or nuclear fractions, which were regulated after ischemic-like stress. GRB2-associated-binding protein 2, which is known to interact with phosphoinositol-3-kinase, was enriched specifically with PTEN-L in the cytosol in proximity to the plasma membrane and their interaction was lost after glutamate exposure. The present study revealed that PTEN and PTEN-L have distinct functions in response to stress and might be involved in different mechanisms of neuroprotection
Antibody binding and functional properties of whey protein hydrolysates obtained under high pressure
7 pages, 4 figures.-- Available online May 18, 2008.This paper examines the potential of high hydrostatic pressure to produce whey protein hydrolysates that combine low immunoglobulin (Ig)G- and IgE-binding with acceptable functional properties, with the aim to produce milk-based ingredients with reduced potential allergenicity that could be used in hypoallergenic foods. Treatment with pepsin and chymotrypsin under high pressure produced, in minutes, hydrolysates in which α-lactalbumin and β-lactoglobulin were totally proteolysed, giving rise to large and hydrophobic peptides. Such hydrolysates presented reduced antigenicity and human IgE-binding properties. The hydrolysates obtained with pepsin at 400 MPa showed improved heat stability, particularly at a pH, close to the isoelectric point of the whey proteins, and their emulsion activity indexes at pH 7.0 were superior to those of the untreated whey proteins. These results suggest that the peptides present retained low antigenicity together with sufficient capacity to form emulsions.This work has been supported by the projects AGL-2004-03322, CONSOLIDER-INGENIO CSD-2007-00063 (Ministerio de Educación
y Ciencia, Spain) and S-0505/AGR/0153 (Comunidad Autónoma de Madrid, Spain).Peer reviewe
Dose response curve shows better survival of TAT.ARC protein transduced primary cortical neurons in comparison to TAT beta galactosidase transduced neurons after oxygen glucose deprivation
Primary mice cortical neuronal cultures on DIV10. OGD for 150 min. Analysis of cell survival after 24h. Exogenous ARC protein
transduction leads to a dose-dependent increase in neuronal survival following
OGD compared to TAT.β-Gal-treated neurons. 4 independent experiments
Delayed administration of TAT.ARC protein attenuates focal ischemic brain injury and fosters recovery in the long-term: behavioral data of neuroscores and rota rod tests, daily temperature and body weight measures
<p>Mice were subjected to 30 min MCAo or sham surgery and TAT.b-Gal or TAT.ARC were administered in the contralateral ventricle after 3h after the onset of ischemia and mice were observed over 28 days. DeSimoni
Neuroscore was performed at indicated time points (Fig. 8) as described (De Simoni et al., 2003; Orsini et al., 2012) with
some modifications. In brief, general health (Table 2) and specific focal
assessments (Table 3) were separately scored, analyzed and finally added to form
a summation score. Summative scores added up to  a maximum of 43 points with more points
meaning more deficits. </p>
<p>Rota rod was
assessed as described recently (Hoffmann et al., 2015) and
the best run out of three replicates at a given time point was used for
statistical analysis. Body temperature was measured daily as survival was documented. </p>
Body temperature was non-invasively assessed at
the same time of the day prior to body weight measurements using subcutaneous transponders
(IPTT-300, Bio Medic Data Systems), for unambiguous identification of mice in
their home cages as described (Kort et al., 1998;
Langer and Fietz, 2014)
Striatal volume and ventricle area was assessed in NeuN DAB sections at 28 days after 30 min MCAo in sham and MCAo mice treated with TAT proteins 3 hours after the onset of ischemia.
Striatal volume and ventricle area was assessed in NeuN DAB sections at 28 days after 30 min tMCAo in sham and MCAo mice treated with TAT proteins 3 hours after the onset of ischemia