Juan Gerez

RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1α during Hypoxia

SUMO conjugation to proteins is involved in the regulation of diverse cellular functions. We have identified a protein, RWD-containing sumoylation enhancer (RSUME), that enhances overall SUMO-1, -2, and -3 conjugation by interacting with the SUMO conjugase Ubc9. RSUME increases noncovalent binding of SUMO-1 to Ubc9 and enhances Ubc9 thioester formation and SUMO polymerization. RSUME enhances the sumoylation of IkB in vitro and in cultured cells, leading to an inhibition of NF-kB transcriptional activity. RSUME is induced by hypoxia and enhances the sumoylation of HIF-1alpha, promoting its stabilization and transcriptional activity during hypoxia. Disruption of the RWD domain structure of RSUME demonstrates that this domain is critical for RSUME action. Together, these findings point to a central role of RSUME in the regulation of sumoylation and, hence, several critical regulatory pathways in mammalian cells.

Host cell entry of respiratory syncytial virus involves macropinocytosis followed by proteolytic activation of the F protein.

Respiratory Syncytial Virus (RSV) is a highly pathogenic member of the Paramyxoviridae that causes severe respiratory tract infections. Reports in the literature have indicated that to infect cells the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. To study the entry process in human tissue culture cells (HeLa, A549), we used fluorescence microscopy and developed quantitative, FACS-based assays to follow virus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. A variety of perturbants were employed to characterize the cellular processes involved. We found that immediately after binding to cells RSV activated a signaling cascade involving the EGF receptor, Cdc42, PAK1, and downstream effectors. This led to a series of dramatic actin rearrangements; the cells rounded up, plasma membrane blebs were formed, and there was a significant increase in fluid uptake. If these effects were inhibited using compounds targeting Na+/H+ exchangers, myosin II, PAK1, and other factors, no infection was observed. The RSV was rapidly and efficiently internalized by an actin-dependent process that had all hallmarks of macropinocytosis. Rather than fusing with the plasma membrane, the viruses thus entered Rab5-positive, fluid-filled macropinosomes, and fused with the membranes of these on the average 50 min after internalization. Rab5 was required for infection. To find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses, we analyzed the fusion protein, F, and could show that, although already cleaved by a furin family protease once, it underwent a second, critical proteolytic cleavage after internalization. This cleavage by a furin-like protease removed a small peptide from the F1 subunits, and made the virus infectious.

Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

In Parkinsons disease and dementia with Lewy bodies, α-synuclein aggregates to form oligomers and fibrils; however, the precise nature of the toxic α-synuclein species remains unclear. A number of synthetic α-synuclein mutations were recently created (E57K and E35K) that produce species of α-synuclein that preferentially form oligomers and increase α-synuclein-mediated toxicity. We have shown that acute lentiviral expression of α-synuclein E57K leads to the degeneration of dopaminergic neurons; however, the effects of chronic expression of oligomer-prone α-synuclein in synapses throughout the brain have not been investigated. Such a study could provide insight into the possible mechanism(s) through which accumulation of α-synuclein oligomers in the synapse leads to neurodegeneration. For this purpose, we compared the patterns of neurodegeneration and synaptic damage between a newly generated mThy-1 α-synuclein E57K transgenic mouse model that is prone to forming oligomers and the mThy-1 α-synuclein wild-type mouse model (Line 61), which accumulates various forms of α-synuclein. Three lines of α-synuclein E57K (Lines 9, 16 and 54) were generated and compared with the wild-type. The α-synuclein E57K Lines 9 and 16 were higher expressings of α-synuclein, similar to α-synuclein wild-type Line 61, and Line 54 was a low expressing of α-synuclein compared to Line 61. By immunoblot analysis, the higher-expressing α-synuclein E57K transgenic mice showed abundant oligomeric, but not fibrillar, α-synuclein whereas lower-expressing mice accumulated monomeric α-synuclein. Monomers, oligomers, and fibrils were present in α-synuclein wild-type Line 61. Immunohistochemical and ultrastructural analyses demonstrated that α-synuclein accumulated in the synapses but not in the neuronal cells bodies, which was different from the α-synuclein wild-type Line 61, which accumulates α-synuclein in the soma. Compared to non-transgenic and lower-expressing mice, the higher-expressing α-synuclein E57K mice displayed synaptic and dendritic loss, reduced levels of synapsin 1 and synaptic vesicles, and behavioural deficits. Similar alterations, but to a lesser extent, were seen in the α-synuclein wild-type mice. Moreover, although the oligomer-prone α-synuclein mice displayed neurodegeneration in the frontal cortex and hippocampus, the α-synuclein wild-type only displayed neuronal loss in the hippocampus. These results support the hypothesis that accumulating oligomeric α-synuclein may mediate early synaptic pathology in Parkinsons disease and dementia with Lewy bodies by disrupting synaptic vesicles. This oligomer-prone model might be useful for evaluating therapies directed at oligomer reduction.

The serine/arginine-rich protein SF2/ASF regulates protein sumoylation

Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF.

RSUME Enhances Glucocorticoid Receptor SUMOylation and Transcriptional Activity

ABSTRACT Glucocorticoid receptor (GR) activity is modulated by posttranslational modifications, including phosphorylation, ubiquitination, and SUMOylation. The GR has three SUMOylation sites: lysine 297 (K297) and K313 in the N-terminal domain (NTD) and K721 within the ligand-binding domain. SUMOylation of the NTD sites mediates the negative effect of the synergy control motifs of GR on promoters with closely spaced GR binding sites. There is scarce evidence on the role of SUMO conjugation to K721 and its impact on GR transcriptional activity. We have previously shown that RSUME (RWD-containing SUMOylation enhancer) increases protein SUMOylation. We now demonstrate that RSUME interacts with the GR and increases its SUMOylation. RSUME regulates GR transcriptional activity and the expression of its endogenous target genes, FKBP51 and S100P. RSUME uncovers a positive role for the third SUMOylation site, K721, on GR-mediated transcription, demonstrating that GR SUMOylation acts positively in the presence of a SUMOylation enhancer. Both mutation of K721 and small interfering RNA-mediated RSUME knockdown diminish GRIP1 coactivator activity. RSUME, whose expression is induced under stress conditions, is a key factor in heat shock-induced GR SUMOylation. These results show that inhibitory and stimulatory SUMO sites are present in the GR and at higher SUMOylation levels the stimulatory one becomes dominant.

Regulation of Pituitary Function by Cytokines

Research performed on the pituitary has proven that cytokines play an important role in maintaining pituitary physiology, affecting not only cell proliferation but also hormone secretion. The effects of cytokines can be autocrine or paracrine. This review gives an overview on the effects of the most studied cytokines in the pituitary. Special interest is focused on interleukin-6 (IL-6) because it has the distinctive characteristic of stimulating pituitary tumor cell growth, but has the opposite effect on normal pituitary cells. On the other hand, IL-6 is a cytokine of interest in the pituitary because recent work has shown that it promotes and maintains senescence in certain types of tumors. Given that the majority of pituitary adenomas are microadenomas and the fact that clinically inapparent pituitary tumors are quite common, senescence, perhaps mediated by IL-6, is an attractive mechanism for explaining the benign nature of pituitary tumors.

RSUME is implicated in HIF-1-induced VEGF-A production in pituitary tumour cells.

The recently cloned small RWD-domain containing protein RSUME was shown to increase protein levels of hypoxia-inducible factor-1α (HIF-1α). The latter is the oxygen-regulated subunit of HIF-1, the most important transcription factor of the cellular adaptive processes to hypoxic conditions. It is also a major regulator of vascular endothelial growth factor-A (VEGF-A), which is critically involved in the complex process of tumour neovascularisation. In this study, the expression and role of RSUME in pituitary tumours was studied. We found that RSUME mRNA was up-regulated in pituitary adenomas and significantly correlated with HIF-1α mRNA levels. Hypoxia (1% O(2)) or treatment with hypoxia-mimicking CoCl(2) enhanced RSUME and HIF-1α expression, induced translocation of HIF-1α to the nuclei and stimulated VEGF-A production both in pituitary tumour cell lines and primary human pituitary adenoma cell cultures. When RSUME expression was specifically down-regulated by siRNA, the CoCl(2)-induced increase VEGF-A secretion was strongly reduced which was shown to be a consequence of the RSUME knockdown-associated reduction of HIF-1α synthesis. Thus, RSUME plays an important role in initiating pituitary tumour neovascularisation through regulating HIF-1α levels and subsequent VEGF-A production and may therefore be critically involved in pituitary adenoma progression.

Preparation and Characterization of Stable α-Synuclein Lipoprotein Particles

Multiple neurodegenerative diseases are caused by the aggregation of the human α-Synuclein (α-Syn) protein. α-Syn possesses high structural plasticity and the capability of interacting with membranes. Both features are not only essential for its physiological function but also play a role in the aggregation process. Recently it has been proposed that α-Syn is able to form lipid-protein particles reminiscent of high-density lipoproteins. Here, we present a method to obtain a stable and homogeneous population of nanometer-sized particles composed of α-Syn and anionic phospholipids. These particles are called α-Syn lipoprotein (nano)particles to indicate their relationship to high-density lipoproteins formed by human apolipoproteins in vivo and of in vitro self-assembling phospholipid bilayer nanodiscs. Structural investigations of the α-Syn lipoprotein particles by circular dichroism (CD) and magic angle solid-state nuclear magnetic resonance (MAS SS-NMR) spectroscopy establish that α-Syn adopts a helical secondary structure within these particles. Based on cryo-electron microscopy (cryo-EM) and dynamic light scattering (DLS) α-Syn lipoprotein particles have a defined size with a diameter of ∼23 nm. Chemical cross-linking in combination with solution-state NMR and multiangle static light scattering (MALS) of α-Syn particles reveal a high-order protein-lipid entity composed of ∼8–10 α-Syn molecules. The close resemblance in size between cross-linked in vitro-derived α-Syn lipoprotein particles and a cross-linked species of endogenous α-Syn from SH-SY5Y human neuroblastoma cells indicates a potential functional relevance of α-Syn lipoprotein nanoparticles.

Intrapituitary Expression and Regulation of the gp130 Cytokine Interleukin-6 and Its Implication in Pituitary Physiology and Pathophysiology

Interleukin (IL)‐6, a member of the gp130 cytokine family, is sometimes designated as an “endocrine” cytokine because of its strong regulatory influence on hormone production. Systemically acting IL‐6 derived from immune cells is a potent stimulator of the hypothalamus–pituitary–adrenal axis and therefore plays an important role in modulating immune–neuroendocrine interactions during inflammatory or infectious processes. However, IL‐6 is also produced within the anterior pituitary by so‐called folliculostellate (FS) cells and is also synthesized in and released by tumor cells in pituitary adenomas. Growth factors (e.g., transforming growth factor‐beta), neuropeptides (e.g., pituitary adenylate cyclase‐activating polypeptide), or hormones (e.g., glucocorticoids) regulate IL‐6 production both in FS and pituitary tumor cells. Interestingly, components of the innate immune system, such as toll‐like receptor 4 and nucleotide‐binding oligomerization domains (NODs), are expressed in FS and pituitary tumor cells. Therefore, cell‐wall components of bacteria (lipopolysaccharide, muramyl dipeptide, diamino pimelic acid) stimulate IL‐6 production in normal and tumoral pituitary. The intrinsic IL‐6 production by FS cells in normal anterior pituitary may participate in immune–neuroendocrine interactions during inflammatory processes. In pituitary adenomas, IL‐6 stimulates hormone secretion, tumor cell proliferation, and the production of angiogenic factors, such as vascular endothelial growth factor‐A, suggesting an important role of IL‐6 in the pathophysiology and progression of pituitary adenomas.

Bone Morphogenetic Protein-4 Control of Pituitary Pathophysiology

Bone morphogenetic protein-4 (BMP-4), a member of the transforming growth factor-Beta(TGF-Beta) family, is overexpressed in different prolactinoma models and induces the development of these lineage adenomas. SMAD proteins activated by growth factors of the TGF-Beta and BMP family interact with estrogen receptors to stimulate the proliferation of prolactin and growth hormone-secreting cells. Furthermore, BMP-4 presents differential expression in normal and adenomatous corticotropes and inhibitory action on corticotropinoma cell proliferation. Moreover, BMP-4 mediates the antiproliferative action of retinoic acid in these cells. The present review highlights not only the crucial and opposite role of BMP-4 in the progression of pituitary adenomas but also that BMP-4 and retinoic acid interaction might serve as a potential new mechanism target for therapeutic approaches for Cushing disease.