Christian Schwartz

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC 2.3.1.87). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN → RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the Km for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

IL-25 and type 2 innate lymphoid cells induce pulmonary fibrosis

Significance Abnormal damage and scarring of tissue (fibrosis) in the lungs can lead to pulmonary fibrosis. Patients that develop the various forms of pulmonary fibrosis are difficult to treat and have a high level of mortality. In this study we have used mouse models to address the role of the cytokine interleukin (IL)-25 and type 2 innate lymphoid cells (ILC2) in pulmonary fibrosis. In animal models we show a role for IL-25 and ILC2 in the generation of pulmonary fibrosis. Furthermore, we have identified elevated levels of IL-25 and ILC2s in the lungs of patients with idiopathic pulmonary fibrosis. This study provides insights on the factors and cells that may initiate pulmonary fibrosis in humans and have therapeutic potential. Disease conditions associated with pulmonary fibrosis are progressive and have a poor long-term prognosis with irreversible changes in airway architecture leading to marked morbidity and mortalities. Using murine models we demonstrate a role for interleukin (IL)-25 in the generation of pulmonary fibrosis. Mechanistically, we identify IL-13 release from type 2 innate lymphoid cells (ILC2) as sufficient to drive collagen deposition in the lungs of challenged mice and suggest this as a potential mechanism through which IL-25 is acting. Additionally, we demonstrate that in human idiopathic pulmonary fibrosis there is increased pulmonary expression of IL-25 and also observe a population ILC2 in the lungs of idiopathic pulmonary fibrosis patients. Collectively, we present an innate mechanism for the generation of pulmonary fibrosis, via IL-25 and ILC2, that occurs independently of T-cell–mediated antigen-specific immune responses. These results suggest the potential of therapeutically targeting IL-25 and ILC2 for the treatment of human fibrotic diseases.

Molecular mechanisms of HIV-1 persistence in the monocyte-macrophage lineage

The introduction of the highly active antiretroviral therapy (HAART) has greatly improved survival. However, these treatments fail to definitively cure the patients and unveil the presence of quiescent HIV-1 reservoirs like cells from monocyte-macrophage lineage. A purge, or at least a significant reduction of these long lived HIV-1 reservoirs will be needed to raise the hope of the viral eradication. This review focuses on the molecular mechanisms responsible for viral persistence in cells of the monocyte-macrophage lineage. Controversy on latency and/or cryptic chronic replication will be specifically evoked. In addition, since HIV-1 infected monocyte-macrophage cells appear to be more resistant to apoptosis, this obstacle to the viral eradication will be discussed. Understanding the intimate mechanisms of HIV-1 persistence is a prerequisite to devise new and original therapies aiming to achieve viral eradication.

An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression.

The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

p21(WAF1) gene promoter is epigenetically silenced by CTIP2 and SUV39H1.

Mainly regulated at the transcriptional level, the cellular cyclin-dependent kinase inhibitor, CDKN1A/p21WAF1 (p21), is a major cell cycle regulator of the response to DNA damage, senescence and tumor suppression. Here, we report that COUP-TF-interacting protein 2 (CTIP2), recruited to the p21 gene promoter, silenced p21 gene transcription through interactions with histone deacetylases and methyltransferases. Importantly, treatment with the specific SUV39H1 inhibitor, chaetocin, repressed histone H3 lysine 9 trimethylation at the p21 gene promoter, stimulated p21 gene expression and induced cell cycle arrest. In addition, CTIP2 and SUV39H1 were recruited to the silenced p21 gene promoter to cooperatively inhibit p21 gene transcription. Induction of p21WAF1 gene upon human immunodeficiency virus 1 (HIV-1) infection benefits viral expression in macrophages. Here, we report that CTIP2 further abolishes Vpr-mediated stimulation of p21, thereby indirectly contributing to HIV-1 latency. Altogether, our results suggest that CTIP2 is a constitutive p21 gene suppressor that cooperates with SUV39H1 and histone methylation to silence the p21 gene transcription.

HIV-1 regulation of latency in the monocyte-macrophage lineage and in CD4+ T lymphocytes

The introduction in 1996 of the HAART raised hopes for the eradication of HIV‐1. Unfortunately, the discovery of latent HIV‐1 reservoirs in CD4+ T cells and in the monocyte‐macrophage lineage proved the optimism to be premature. The long‐lived HIV‐1 reservoirs constitute a major obstacle to the eradication of HIV‐1. In this review, we focus on the establishment and maintenance of HIV‐1 latency in the two major targets for HIV‐1: the CD4+ T cells and the monocyte‐macrophage lineage. Understanding the cell‐type molecular mechanisms of establishment, maintenance, and reactivation of HIV‐1 latency in these reservoirs is crucial for efficient therapeutic intervention. A complete viral eradication, the holy graal for clinicians, might be achieved by strategic interventions targeting latently and productively infected cells. We suggest that new approaches, such as the combination of different kinds of proviral activators, may help to reduce dramatically the size of latent HIV‐1 reservoirs in patients on HAART.

Melatonin synthesis: arylalkylamine N-acetyltransferases in trout retina and pineal organ are different.

Serotonin N-acetyltransferase (AANAT) is the first enzyme in the conversion of serotonin to melatonin. Changes in AANAT activity determine the daily rhythm in melatonin secretion. Two AANAT genes have been identified in the pike, pAANAT-1 and pAANAT-2, expressed in the retina and in the pineal, respectively. The genes preferentially expressed in these tissues encode proteins with distinctly different kinetic characteristics. Like the pike, trout retina primarily expresses the AANAT-1 gene and trout pineal primarily expresses the AANAT-2 gene. Here we show that the kinetic characteristics of AANAT in these tissues differ as in pike. These differences include optimal temperature for activity (pineal: 12°C; retina: 25°C) and relative affinity for indoleethylamines compared to phenylethylamines. In addition, retinal AANAT exhibited substrate inhibition, which was not seen with pineal AANAT. The kinetic differences between AANAT-1 and AANAT-2 appear to be defining characteristics of these gene subfamilies, and are not species specific.

Functional interactions between C/EBP, Sp1, and COUP-TF regulate human immunodeficiency virus type 1 gene transcription in human brain cells.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infects the central nervous system (CNS) and plays a direct role in the pathogenesis of AIDS dementia. However, mechanisms underlying HIV-1 gene expression in the CNS are poorly understood. The importance of CCAAT/enhancer binding proteins (C/EBP) for HIV-1 expression in cells of the immune system has been recently reported. In this study, we have examined the role and the molecular mechanisms by which proteins of the C/EBP family regulate HIV-1 gene transcription in human brain cells. We found that NF-IL6 acts as a potent activator of the long terminal repeat (LTR)-driven transcription in microglial and oligodendroglioma cells. In contrast, C/EBPγ inhibits NF-IL6-induced activation. Consistent with previous data, our transient expression results show cell-type-specific NF-IL6-mediated transactivation. In glial cells, full activation needs the presence of the C/EBP binding sites; however, NF-IL6 is still able to function via the minimal −40/+80 region. In microglial cells, C/EBP sites are not essential, since NF-IL6 acts through the −68/+80 LTR region, containing two binding sites for the transcription factor Sp1. Moreover, we show that functional interactions between NF-IL6 and Sp1 lead to synergistic transcriptional activation of the LTR in oligodendroglioma and to mutual repression in microglial cells. We further demonstrate that NF-IL6 physically interacts with the nuclear receptor chicken ovalbumin upstream promoter transcription factor (COUP-TF), via its DNA binding domain, in vitro and in cells, which results in mutual transcriptional repression. These findings reveal how the interplay of NF-IL6 and C/EBPγ, together with Sp1 and COUP-TF, regulates HIV-1 gene transcription in brain cells.

CTIP2 is a negative regulator of P-TEFb.

The positive transcription elongation factor b (P-TEFb) is involved in physiological and pathological events including inflammation, cancer, AIDS, and cardiac hypertrophy. The balance between its active and inactive form is tightly controlled to ensure cellular integrity. We report that the transcriptional repressor CTIP2 is a major modulator of P-TEFb activity. CTIP2 copurifies and interacts with an inactive P-TEFb complex containing the 7SK snRNA and HEXIM1. CTIP2 associates directly with HEXIM1 and, via the loop 2 of the 7SK snRNA, with P-TEFb. In this nucleoprotein complex, CTIP2 significantly represses the Cdk9 kinase activity of P-TEFb. Accordingly, we show that CTIP2 inhibits large sets of P-TEFb- and 7SK snRNA-sensitive genes. In hearts of hypertrophic cardiomyopathic mice, CTIP2 controls P-TEFb-sensitive pathways involved in the establishment of this pathology. Overexpression of the β-myosin heavy chain protein contributes to the pathological cardiac wall thickening. The inactive P-TEFb complex associates with CTIP2 at the MYH7 gene promoter to repress its activity. Taken together, our results strongly suggest that CTIP2 controls P-TEFb function in physiological and pathological conditions.

Achieving a cure for HIV infection: do we have reasons to be optimistic?

The introduction of highly active antiretroviral therapy (HAART) in 1996 has transformed a lethal disease to a chronic pathology with a dramatic decrease in mortality and morbidity of AIDS-related symptoms in infected patients. However, HAART has not allowed the cure of HIV infection, the main obstacle to HIV eradication being the existence of quiescent reservoirs. Several other problems have been encountered with HAART (such as side effects, adherence to medication, emergence of resistance and cost of treatment), and these motivate the search for new ways to treat these patients. Recent advances hold promise for the ultimate cure of HIV infection, which is the topic of this review. Besides these new strategies aiming to eliminate the virus, efforts must be made to improve current HAART. We believe that the cure of HIV infection will not be attained in the short term and that a strategy based on purging the reservoirs has to be associated with an aggressive HAART strategy.