Search

Activating transcription factor-5 (ATF5) is a stress-response transcription factor induced upon different cell stressors like fasting, amino-acid limitation, cadmium or arsenite. ATF5 is also induced, and promotes transcription of anti-apoptotic target genes like MCL1, during the unfolded protein response (UPR) triggered by endoplasmic reticulum stress. In the brain, high ATF5 levels are found in gliomas and also in neural progenitor cells, which need to decrease their ATF5 levels for differentiation into mature neurons or glia. This initially led to believe that ATF5 is not expressed in adult neurons. More recently, we reported basal neuronal ATF5 expression in adult mouse brain and its neuroprotective induction during UPR in a mouse model of status epilepticus. Here we aimed to explore whether ATF5 is also expressed by neurons in human brain both in basal conditions and in Huntington’s disease (HD), where UPR has been described to be partially impaired due to defective ATF6 processing. Apart from confirming that ATF5 is present in human adult neurons, here we report accumulation of ATF5 within the characteristic polyglutamine-containing neuronal nuclear inclusions in brains of HD patients and mice. This correlates with decreased levels of soluble ATF5 and of its antiapoptotic target MCL1. We then confirmed the deleterious effect of ATF5 deficiency in a Caenorhabditis elegans model of polyglutamine-induced toxicity. Finally, ATF5 overexpression attenuated polyglutamine-induced apoptosis in a cell model of HD. These results reflect that decreased ATF5 in HD—probably secondary to sequestration into inclusions—renders neurons more vulnerable to mutant huntingtin-induced apoptosis and that ATF5-increasing interventions might have therapeutic potential for HD., CiberNed-ISCIII collaborative Grants PI2013/09-2 and PI2015-2/06-3 and by grants from Spanish Ministry of Economy and Competitiveness (MINECO): SAF2009-08233 and SAF2015-65371-R to JJL, by Fundación BBVA and by Fundación Ramón Areces

Molecular chaperones are a group of proteins that assist in protein homeostasis. They not only prevent protein misfolding and aggregation, but also target misfolded proteins for degradation. Despite differences in structure, all types of chaperones share a common general feature, a surface that recognizes and interacts with the misfolded protein. This and other, more specialized properties can be adapted for various nanotechnological purposes, by modification of the original biomolecules or by de novo design based on artificial structures.

The INhibitor of Growth (ING) family of tumor suppressors regulates the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at position K4 (H3K4me3). This modification is recognized by the plant homeodomain (PHD) present at the C-terminus in the five members of the ING family. ING4 facilitates histone H3 acetylation by the HBO1 complex. Here, we show that ING4 forms homodimers through its N-terminal domain, which folds independently into an elongated coiled-coil structure. The central region of ING4, which contains the nuclear localization sequence, is disordered and flexible and does not directly interact with p53, or does it with very low affinity, in contrast to previous findings. The NMR analysis of the full-length protein reveals that the two PHD fingers of the dimer are chemically equivalent and independent of the rest of the molecule. The detailed NMR analysis of the full-length dimeric protein binding to histone H3K4me3 shows essentially the same binding site and affinity as the isolated PHD finger. Therefore, the ING4 dimer has two identical and independent binding sites for H3K4me3 tails, which, in the context of the chromatin, could belong to the same or to different nucleosomes. These results show that ING4 is a bivalent reader of the chromatin H3K4me3 modification and suggest a mechanism for enhanced targeting of the HBO1 complex to specific chromatin sites. This mechanism could be common to other ING-containing remodeling complexes. © 2009 Elsevier Ltd., This work was supported by grants from Ministerio de Ciencia e Innovación (CTQ2008-03115/BQU), European Union (3D-REPERTOIRE, contract no. LSHG-CT-2005-512028), Fundación Mutua Madrileña to F.J.B., and ETORTEK-2008 grant to CIC bioGUNE. We also acknowledge the support to European Molecular Biology Laboratory Hamburg by European Commission–Research Infrastructure Action contract no. RII/2004/5060008. P.B. holds a Ramón y Cajal contract, and M.R.F.-F. was a Career Development Fellow of the MRC-UK.

PEPSCAN analysis has been used to characterize the immunogenic regions of the capsid protein (CP) in virions of plum pox potyvirus (PPV). In addition to the well-known highly immunogenic N- and C-terminal domains of CP, regions within the core domain of the protein have also shown high immunogenicity. Moreover, the N terminus of CP is not homogeneously immunogenic, alternatively showing regions frequently recognized by antibodies and others that are not recognized at all. These results have helped us to design efficient antigen presentation vectors based on PPV. As predicted by PEPSCAN analysis, a small displacement of the insertion site in a previously constructed vector, PPV-γ, turned the derived chimeras into efficient immunogens. Vectors expressing foreign peptides at different positions within a highly immunogenic region (amino acids 43 to 52) in the N-terminal domain of CP were the most effective at inducing specific antibody responses against the foreign sequence.

Activating transcription factor 5 (ATF5) is a basic-leucine-zipper transcription factor of the ATF/CREB family. The Atf5 gene generates two transcripts, Atf5α and Atf5β, of which Atf5α is known to be selectively translated upon endoplasmic reticulum stress response in non-neuronal cells. ATF5 is highly expressed in the developing brain where it modulates proliferation of neural progenitor cells. These cells show a high level of ATF5 that has to decrease to allow them to differentiate into mature neurons or glial cells. This has led to the extended notion that differentiated neural cells do not express ATF5 unless they undergo tumourigenic transformation. However, no systematic analysis of the distribution of ATF5 in adult brain or of its potential role in neuronal endoplasmic reticulum stress response has been reported. By immunostaining here we confirm highest ATF5 levels in neuroprogenitor cells of the embryonic and adult subventricular zone but also found ATF5 in a large variety of neurons in adult mouse brain. By combining Atf5 in situ hybridization and immunohistochemistry for the neuronal marker NeuN we further confirmed Atf5 messenger RNA in adult mouse neurons. Quantitative reverse transcriptase polymerase chain reaction demonstrated that Atf5α is the most abundant transcript in adult mouse encephalon and injection of the endoplasmic reticulum stress inducer tunicamycin into adult mouse brain increased neuronal ATF5 levels. Accordingly, ATF5 levels increased in hippocampal neurons of a mouse model of status epilepticus triggered by intra-amygdala injection of kainic acid, which leads to abnormal hippocampal neuronal activity and endoplasmic reticulum stress. Interestingly, ATF5 upregulation occurred mainly in hippocampal neuronal fields that do not undergo apoptosis in this status epilepticus model such as CA1 and dentate gyrus, thus suggesting a neuroprotective role. This was confirmed in a primary neuronal culture model in which ATF5 overexpression resulted in decreased endoplasmic reticulum stress-induced apoptosis and the opposite result was achieved by Atf5 RNA interference. Furthermore, in vivo administration of the eIF2α phosphatase inhibitor salubrinal resulted in increased ATF5 hippocampal levels and attenuated status epilepticus-induced neuronal death in the vulnerable CA3 subfield. In good agreement with the neuroprotective effect of increased ATF5, we found that apoptosis-resistant epileptogenic foci from patients with temporal lobe epilepsy also showed increased levels of ATF5. Thus, our results demonstrate that adult neurons express ATF5 and that they increase its levels upon endoplasmic reticulum stress as a pro-survival mechanism, thus opening a new field for neuroprotective strategies focused on ATF5 modulation. © 2013 The Author (2013).

Plum pox virus (PPV) is a member of the Potyvirus genus of plant viruses. Labeling with UDP-[3H]galactose and galactosyltransferase indicated that the capsid protein (CP) of PPV is a glycoprotein with N-acetylglucosamine terminal residues. Mass spectrometry analysis of different PPV isolates and mutants revealed O-linked N-acetylglucosamination, a modification barely studied in plant proteins, of serine and/or threonine residues near the amino end of PPV CP. CP of PPV virions is also modified by serine and threonine phosphorylation, as shown by Western blot analysis with anti-phosphoserine and anti-phosphothreonine antibodies. Thus, "yin-yang" glycosylation and phosphorylation may play an important role in the regulation of the different functions in which the potyviral CP is involved.

The development of an antigen presentation system based on the plum pox potyvirus (PPV) is here described. The amino-terminal part of PPV capsid protein was chosen as the site for expression of foreign antigenic peptides. Modifications in this site were engineered to avoid the capability of natural transmission by aphids of this PPV vector. As a first practical attempt, different forms of an antigenic peptide (single and tandem repetition) from the VP2 capsid protein of canine parvovirus (CPV) were expressed. Both chimeras are able to infect Nicotiana clevelandii plants with similar characteristics to wild-type virus and remain genetically stable after several plant passages. The antigenicity of purified chimeric virions was demonstrated, proving the suitability of this system for diagnostic purposes. Moreover, mice and rabbits immunized with chimeric virions developed CPV-specific antibodies, which showed neutralizing activity.