José González-Santamaría

Kaposi's Sarcoma-Associated Herpesvirus Protein LANA2 Disrupts PML Oncogenic Domains and Inhibits PML-Mediated Transcriptional Repression of the Survivin Gene

ABSTRACT Infection by herpesviruses causes a dramatic disturbance of PML oncogenic domains (PODs) that has been suggested to be essential for viral lytic replication. Several proteins from Kaposis sarcoma-associated herpesvirus (KSHV) have been tested as putative POD-disrupting factors with negative results. Here, we show that LANA2, a viral protein that is absolutely required for the viability and proliferation of KSHV-infected primary effusion lymphoma (PEL) cells, increases the levels of SUMO2-ubiquitin-modified PML and induces the disruption of PODs by a proteasome-mediated mechanism. In addition, we demonstrate that this disruption is largely dependent on both the integrity of a SUMO interaction motif in LANA2 and the lysine 160 from PML. Moreover, silencing of LANA2 expression in PEL cells by RNA interference led to an increase in the PML levels. Finally, we demonstrate that LANA2 relieves PML-mediated transcriptional repression of survivin, a protein that directly contributes to malignant progression of PEL. This represents the first example of inactivation of these important antiviral structures by KSHV.

SIRT1 Stabilizes PML promoting its Sumoylation

SIRT1, the closest mammalian homolog of yeast Sir2, is an NAD+-dependent deacetylase with relevant functions in cancer, aging, and metabolism among other processes. SIRT1 has a diffuse nuclear localization but is recruited to the PML nuclear bodies (PML-NBs) after PML upregulation. However, the functions of SIRT1 in the PML-NBs are unknown. In this study we show that primary mouse embryo fibroblasts lacking SIRT1 contain reduced PML protein levels that are increased after reintroduction of SIRT1. In addition, overexpression of SIRT1 in HEK-293 cells increases the amount of PML protein whereas knockdown of SIRT1 reduces the size and number of PML-NBs and the levels of PML protein in HeLa cells. SIRT1 stimulates PML sumoylation in vitro and in vivo in a deacetylase-independent manner. Importantly, the absence of SIRT1 reduces the apoptotic response of vesicular stomatitis virus-infected cells and favors the extent of this PML-sensitive virus replication. These results show a novel function of SIRT1 in the control of PML and PML-NBs.

Regulation of the tumor suppressor PTEN by SUMO

The crucial function of the PTEN tumor suppressor in multiple cellular processes suggests that its activity must be tightly controlled. Both, membrane association and a variety of post-translational modifications, such as acetylation, phosphorylation, and mono- and polyubiquitination, have been reported to regulate PTEN activity. Here, we demonstrated that PTEN is also post-translationally modified by the small ubiquitin-like proteins, small ubiquitin-related modifier 1 (SUMO1) and SUMO2. We identified lysine residue 266 and the major monoubiquitination site 289, both located within the C2 domain required for PTEN membrane association, as SUMO acceptors in PTEN. We demonstrated the existence of a crosstalk between PTEN SUMOylation and ubiquitination, with PTEN-SUMO1 showing a reduced capacity to form covalent interactions with monoubiquitin and accumulation of PTEN-SUMO2 conjugates after inhibition of the proteasome. Moreover, we found that virus infection induces PTEN SUMOylation and favors PTEN localization at the cell membrane. Finally, we demonstrated that SUMOylation contributes to the control of virus infection by PTEN.

LOXL4 Is Induced by Transforming Growth Factor β1 through Smad and JunB/Fra2 and Contributes to Vascular Matrix Remodeling

ABSTRACT Transforming growth factor β1 (TGF-β1) is a pleiotropic factor involved in the regulation of extracellular matrix (ECM) synthesis and remodeling. In search for novel genes mediating the action of TGF-β1 on vascular ECM, we identified the member of the lysyl oxidase family of matrix-remodeling enzymes, lysyl oxidase-like 4 (LOXL4), as a direct target of TGF-β1 in aortic endothelial cells, and we dissected the molecular mechanism of its induction. Deletion mapping and mutagenesis analysis of the LOXL4 promoter demonstrated the absolute requirement of a distal enhancer containing an activator protein 1 (AP-1) site and a Smad binding element for TGF-β1 to induce LOXL4 expression. Functional cooperation between Smad proteins and the AP-1 complex composed of JunB/Fra2 accounted for the action of TGF-β1, which involved the extracellular signal-regulated kinase (ERK)-dependent phosphorylation of Fra2. We furthermore provide evidence that LOXL4 was extracellularly secreted and significantly contributed to ECM deposition and assembly. These results suggest that TGF-β1-dependent expression of LOXL4 plays a role in vascular ECM homeostasis, contributing to vascular processes associated with ECM remodeling and fibrosis.

The profibrotic role of endothelin-1: is the door still open for the treatment of fibrotic diseases?

The endothelin (ET) system consists of two G-protein-coupled receptors (ETA and ETB), three peptide ligands (ET-1, ET-2 and ET-3), and two activating peptidases (endothelin-converting enzyme-, ECE-1 and ECE-2). While initially described as a vasoregulatory factor, shown to influence several cardiovascular diseases, from hypertension to heart failure, ET-1, the predominant form in most cells and tissues, has expanded its pathophysiological relevance by recent evidences implicating this factor in the regulation of fibrosis. In this article, we review the current knowledge of the role of ET-1 in the development of fibrosis, with particular focus on the regulation of its biosynthesis and the molecular mechanisms involved in its profibrotic actions. We summarize also the contribution of ET-1 to fibrotic disorders in several organs and tissues. The development and availability of specific ET receptor antagonists have greatly stimulated a number of clinical trials in these pathologies that unfortunately have so far given negative or inconclusive results. This review finally discusses the circumstances underlying these disappointing results, as well as provides basic and clinical researchers with arguments to keep exploring the complex physiology of ET-1 and its therapeutic potential in the process of fibrosis.

Matrix cross-linking lysyl oxidases are induced in response to myocardial infarction and promote cardiac dysfunction

AIMS After myocardial infarction (MI), extensive remodelling of the extracellular matrix contributes to scar formation. While aiming to preserve tissue integrity, this fibrotic response is also associated with adverse events, including a markedly increased risk of heart failure, ventricular arrhythmias, and sudden cardiac death. Cardiac fibrosis is characterized by extensive deposition of collagen and also by increased stiffness as a consequence of enhanced collagen cross-linking. Members of the lysyl oxidase (LOX) family of enzymes are responsible for the formation of collagen cross-links. This study investigates the contribution of LOX family members to the heart response to MI. METHODS AND RESULTS Experimental MI was induced in C57BL/6 mice by permanent ligation of the left anterior descending coronary artery. The expression of LOX isoforms (LOX and LOXL1-4) was strongly increased upon MI, and this response was accompanied by a significant accumulation of mature collagen fibres in the infarcted area. LOX expression was observed in areas of extensive remodelling, partially overlapping with α-smooth muscle actin-expressing myofibroblasts. Tumour growth factor-β as well as hypoxia-activated pathways contributed to the induction of LOX expression in cardiac fibroblasts. Finally, in vivo post-infarction treatment with the broadband LOX inhibitor β-aminopropionitrile or, selectively, with a neutralizing antibody against the canonical LOX isoform attenuated collagen accumulation and maturation and also resulted in reduced ventricular dilatation and improved cardiac function. CONCLUSION LOX family members contribute significantly to the detrimental effects of cardiac remodelling, highlighting LOX inhibition as a potential therapeutic strategy for post-infarction recovery.

Covalent modification by SUMO is required for efficient disruption of PML oncogenic domains by Kaposi's sarcoma-associated herpesvirus latent protein LANA2

The multifunctional Kaposis sarcoma-associated herpesvirus (KSHV) latent protein latency-associated nuclear antigen 2 (LANA2) has a critical role in KSHV-induced B-cell malignancies. LANA2 increases the level of small ubiquitin-like modifier (SUMO)2-ubiquitin-modified PML and induces the disruption of PML oncogenic domains (PODs) by a process that requires a non-covalent SUMO interaction domain (SIM) in LANA2. We now demonstrate that LANA2 is covalently conjugated to SUMO1 and SUMO2 both in vitro and in latently KSHV-infected B-cells. We show that a LANA2 SIM mutant exhibits a slightly altered sumoylation pattern, which suggests that non-covalent SUMO interactions represent a mechanism for determining SUMO substrate recognition and modification. In addition, several lysine residues were mapped as SUMO conjugation sites. A sumoylation-deficient mutant shows impaired ability to induce disruption of PODs, which suggests that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interaction between LANA2 and other SUMO-modified or SUMO-interacting proteins required for disruption of PODs.

Acetylation is indispensable for p53 antiviral activity.

Tumor suppressor p53 is known to be a direct transcriptional target of type I interferons (IFNs), contributing to virus-induced apoptosis, and in turn activating itself the interferon pathway. Acetylation, among many other post-translational modifications of p53, is thought to exert a crucial role regulating p53 activity. Here, we examined the contribution of this modification on the antiviral activity mediated by p53. Our results show that virus infection induces p53 acetylation at lysine 379, and that this modification is absolutely required for p53-dependent transcriptional transactivation of both, pro-apoptotic and IFN-stimulated genes induced by virus infection, and for p53-mediated control of virus replication. Thus, our study identifies p53 acetylation as an indispensable event that enables the p53-mediated antiviral response.

Activation of NF-kB Pathway by Virus Infection Requires Rb Expression

The retinoblastoma protein Rb is a tumor suppressor involved in cell cycle control, differentiation, and inhibition of oncogenic transformation. Besides these roles, additional functions in the control of immune response have been suggested. In the present study we investigated the consequences of loss of Rb in viral infection. Here we show that virus replication is increased by the absence of Rb, and that Rb is required for the activation of the NF-kB pathway in response to virus infection. These results reveal a novel role for tumor suppressor Rb in viral infection surveillance and further extend the concept of a link between tumor suppressors and antiviral activity.

Regulation of Vaccinia Virus E3 Protein by Small Ubiquitin-Like Modifier Proteins

ABSTRACT The vaccinia virus (VACV) E3 protein is essential for virulence and has antiapoptotic activity and the ability to impair the host innate immune response. Here we demonstrate that E3 interacts with SUMO1 through a small ubiquitin-like modifier (SUMO)-interacting motif (SIM). SIM integrity is required for maintaining the stability of the viral protein and for the covalent conjugation of E3 to SUMO1 or SUMO2, a modification that has a negative effect on the E3 transcriptional transactivation of the p53-upregulated modulator of apoptosis (PUMA) and APAF-1 genes. We also demonstrate that E3 is ubiquitinated, a modification that does not destabilize the wild-type protein but triggers the degradation of an E3-ΔSIM mutant. This report constitutes the first demonstration of the important roles that both SUMO and ubiquitin play in the regulation of the VACV protein E3.