Maria Ines Gutierrez

Acetylation of HIV-1 integrase by p300 regulates viral integration

Integration of HIV‐1 into the human genome, which is catalyzed by the viral protein integrase (IN), preferentially occurs near transcriptionally active genes. Here we show that p300, a cellular acetyltransferase that regulates chromatin conformation through the acetylation of histones, also acetylates IN and controls its activity. We have found that p300 directly binds IN both in vitro and in the cells, as also specifically demonstrated by fluorescence resonance energy transfer technique analysis. This interaction results in the acetylation of three specific lysines (K264, K266, K273) in the carboxy‐terminus of IN, a region that is required for DNA binding. Acetylation increases IN affinity to DNA, and promotes the DNA strand transfer activity of the protein. In the context of the viral replication cycle, point mutations in the IN acetylation sites abolish virus replication by specifically impairing its integration capacity. This is the first demonstration that HIV‐1 IN activity is specifically regulated by post‐translational modification.

Recurrent DNA inversion rearrangements in the human genome

Several lines of evidence suggest that reiterated sequences in the human genome are targets for nonallelic homologous recombination (NAHR), which facilitates genomic rearrangements. We have used a PCR-based approach to identify breakpoint regions of rearranged structures in the human genome. In particular, we have identified intrachromosomal identical repeats that are located in reverse orientation, which may lead to chromosomal inversions. A bioinformatic workflow pathway to select appropriate regions for analysis was developed. Three such regions overlapping with known human genes, located on chromosomes 3, 15, and 19, were analyzed. The relative proportion of wild-type to rearranged structures was determined in DNA samples from blood obtained from different, unrelated individuals. The results obtained indicate that recurrent genomic rearrangements occur at relatively high frequency in somatic cells. Interestingly, the rearrangements studied were significantly more abundant in adults than in newborn individuals, suggesting that such DNA rearrangements might start to appear during embryogenesis or fetal life and continue to accumulate after birth. The relevance of our results in regard to human genomic variation is discussed.

Concerted action of cellular JNK and Pin1 restricts HIV-1 genome integration to activated CD4+ T lymphocytes

Long-standing evidence indicates that quiescent human peripheral blood T lymphocytes (PBLs) do not support efficient HIV infection. In resting PBLs, reverse transcription of viral RNA takes longer than in activated cells, partially because formation of the late products of reverse transcription is decreased by RNA binding by apolipoprotein B mRNA–editing enzyme, catalytic polypeptide-like 3G (APOBEC3G). In a subsequent step, integration of the viral complementary DNA that is eventually formed is markedly impaired. Here we show that cellular c-Jun N-terminal kinase (JNK), an enzyme that is not expressed in resting CD4+ T cells, regulates permissiveness to HIV-1 infection, and we unravel a new, sequential post-translational pathway of protein modification that regulates viral DNA integration. We found that, in activated T lymphocytes, viral integrase, which mediates HIV-1 cDNA integration into the host cell genome, is phosphorylated by JNK on a highly conserved serine residue in its core domain. Phosphorylated integrase, in turn, becomes a substrate for the cellular peptidyl prolyl-isomerase enzyme Pin1, which catalyzes a conformational modification of integrase. These concerted activities increase integrase stability and are required for efficient HIV-1 integration and infection. Lack of these modifications restricts viral infection in nonactivated, primary CD4+ T lymphocytes.

Identification of Specific Molecular Structures of Human Immunodeficiency Virus Type 1 Tat Relevant for Its Biological Effects on Vascular Endothelial Cells

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Tat transactivates viral genes and is released by infected cells, acting as a soluble mediator. In endothelial cells (EC), it activates a proangiogenic program by activating vascular endothelial growth factor receptor type 2 (VEGFR-2) and integrins. A structure-activity relationship study was performed by functional analysis of Tat substitution and deletion variants to define the Tat determinants necessary for EC activation. Variants were made (i) in the basic and (ii) in the cysteine-rich domains and (iii) in the C-terminal region containing the RGD sequence required for integrin recognition. Our results led to the following conclusions. (i) Besides a high-affinity binding site corresponding to VEGFR-2, EC express low-affinity binding sites. (ii) The basic and the cysteine-rich variants bind only to the low-affinity binding sites and do not promote tyrosine phosphorylation of VEGFR-2. Furthermore, they have a reduced ability to activate EC in vitro, and they lack angiogenic activity. (iii) Mutants with mutations in the C-terminal region are partially defective for in vitro biological activities and in vivo angiogenesis, but they activate VEGFR-2 as Tat wild type. In conclusion, regions encoded by the first exon of tat are necessary and sufficient for activation of VEGFR-2. However, the C-terminal region, most probably through RGD-mediated integrin engagement, is indispensable for full activation of an in vitro and in vivo angiogenic program.

Regulation of E2F-1 after DNA Damage by p300-Mediated Acetylation and Ubiquitination

Here we report a novel, non-competitive mechanism that links acetylation and ubiquitination, in which the association of transcription factor E2F-1 with the cellular co-activator and acetyltransferase p300 determines its acetylation and subsequent ubiquitination. By using an antibody specifically recognizing the acetylated form of E2F-1 (AcE2F-1), we found that, after DNA damage, AcE2F-1 accumulates in the cells in a time-dependent manner, and that acetylation is increased by the expression of p300. Remarkably, the same DNA damaging conditions also induce the accumulation of ubiquitinated E2F-1, an event that is again markedly stimulated by p300 overexpression. The effects of p300 on E2F-1 ubiquitination require the integrity of the HAT domain of p300 and of the three acetylated lysines in E2F-1. Of note, p300-induced E2F-1 ubiquitination does not depend on the p45Skp2 E3 ligase, since it does not extend to other p45Skp2 targets and also occurs with an E2F-1 mutant devoid of the p45Skp2-binding domain but still retaining the acetylated region. Finally, p300-induced E2F-1 ubiquitination is not influenced by RB.

Reversible acetylation regulates vascular endothelial growth factor receptor-2 activity

The tyrosine kinase receptor vascular endothelial growth factor receptor 2 (VEGFR2) is a key regulator of angiogenesis. Here we show that VEGFR2 is acetylated in endothelial cells both at four lysine residues forming a dense cluster in the kinase insert domain and at a single lysine located in the receptor activation loop. These modifications are under dynamic control of the acetyltransferase p300 and two deacetylases HDAC5 and HDAC6. We demonstrate that VEGFR2 acetylation essentially regulates receptor phosphorylation. In particular, VEGFR2 acetylation significantly alters the kinetics of receptor phosphorylation after ligand binding, allowing receptor phosphorylation and intracellular signaling upon prolonged stimulation with VEGF. Molecular dynamics simulations indicate that acetylation of the lysine in the activation loop contributes to the transition to an open active state, in which tyrosine phosphorylation is favored by better exposure of the kinase target residues. These findings indicate that post-translational modification by acetylation is a critical mechanism that directly affects VEGFR2 function.

A protein target site in an early replicated human DNA sequence: a highly conserved binding motif.

We have previously reported that a human nuclear factor, probably corresponding to the USF/MLTF protein [1,2], is able to bind specifically to a DNA sequence present in DNA replicated at the onset of S-phase [3]. Here we demonstrate that the same factor binds also to several other similar sequences, present in eukaryotic and viral genomes. Mutations or methylation in a CpG dinucleotide, central in the palindromic binding site, completely abolish binding. Furthermore, we present evidence for the existence of at least two other nuclear proteins in human cells with the same DNA binding specificity. The data presented suggest a strong evolutionary conservation, among distantly related organisms, of the binding motif, which is probably the target of a number of nuclear factors that share the same DNA binding specificity albeit in the context of different functions.

In vitro selection of HIV-1 TAR variants by the Tat protein.

Starting from a pool of 10(13) RNA sequences, we isolated a number of TAR RNA variants after nine rounds of selection by binding to recombinant Tat in vitro (SELEX procedure). Sequence analysis of part of the selected molecular species indicated that two TAR variants (clones A and B) were, respectively, represented five and four times. These two groups of sequences constituted approximately 25% of the total number of analyzed clones (9/34). As far as the primary and presumptive secondary structures of the wild-type TAR are concerned, the selected A and B variants showed an almost complete sequence conservation of the Tat-binding domain, but the configuration of this nucleotide region differed within the secondary structure. Despite this difference, as verified by gel retardation and filter binding assays, both the A and B variants bound Tat in vitro with an affinity that was very close to that of the wild-type TAR. Conversely, neither variant sustained Tat-mediated trans-activation in vivo when they replaced the wild-type TAR inside the long terminal repeat of HIV_1. Taken together, our results suggest that these TAR variants have lost the ability to bind cell factor(s) in vivo and may therefore represent useful decoys for the inhibition of HIV-1 replication.

Paracrine effect of regulatory T cells promotes cardiomyocyte proliferation during pregnancy and after myocardial infarction

Cardiomyocyte proliferation stops at birth when the heart is no longer exposed to maternal blood and, likewise, to regulatory T cells (Tregs) that are expanded to promote maternal tolerance towards the fetus. Here, we report a role of Tregs in promoting cardiomyocyte proliferation. Treg-conditioned medium promotes cardiomyocyte proliferation, similar to the serum from pregnant animals. Proliferative cardiomyocytes are detected in the heart of pregnant mothers, and Treg depletion during pregnancy decreases both maternal and fetal cardiomyocyte proliferation. Treg depletion after myocardial infarction results in depressed cardiac function, massive inflammation, and scarce collagen deposition. In contrast, Treg injection reduces infarct size, preserves contractility, and increases the number of proliferating cardiomyocytes. The overexpression of six factors secreted by Tregs (Cst7, Tnfsf11, Il33, Fgl2, Matn2, and Igf2) reproduces the therapeutic effect. In conclusion, Tregs promote fetal and maternal cardiomyocyte proliferation in a paracrine manner and improve the outcome of myocardial infarction.Regulatory T cells (Tregs) expand during pregnancy to promote tolerance towards the fetus. Here the authors show that Tregs induce proliferation of fetal and maternal cardiomyocytes during pregnancy and enhance myocardial repair via proliferation-promoting paracrine actions.

Reversible Notch1 acetylation tunes proliferative signalling in cardiomyocytes

Aims The Notch signalling pathway regulates the balance between proliferation and differentiation in several tissues, including the heart. Our previous work has demonstrated that the proliferative potential of neonatal cardiomyocytes relies on Notch1 activity. A deep investigation on the biochemical regulation of the Notch signalling in cardiomyocytes is the focus of the current research. Methods and results We show that the Notch1 intracellular domain is acetylated in proliferating neonatal rat cardiomyocytes and that acetylation tightly controls the amplitude and duration of Notch signalling. We found that acetylation extends the half-life of the protein, and enhanced its transcriptional activity, therefore counteracting apoptosis and sustaining cardiomyocyte proliferation. Sirt1 acted as a negative modulator of Notch1 signalling; its overexpression in cardiomyocytes reverted Notch acetylation and dampened its stability. A constitutively acetylated fusion protein between Notch1 and the acetyltransferase domain of p300 promoted cardiomyocyte proliferation, which was remarkably sustained over time. Viral vector-mediated expression of this protein enhanced heart regeneration after apical resection in neonatal mice. Conclusion These results identify the reversible acetylation of Notch1 as a novel mechanism to modulate its signalling in the heart and tune the proliferative potential of cardiomyocytes.