SIRT6 stabilization and cytoplasmic localization in macrophages regulates acute and chronic inflammation in mice

Acute and chronic inflammations are key homeostatic events in health and disease. Sirtuins (SIRTs), a family of NAD-dependent protein deacylases, play a pivotal role in the regulation of these inflammatory responses. Indeed, SIRTs have anti-inflammatory effects through a myriad of signaling cascades, including histone deacetylation and gene silencing, p65/RelA deacetylation and inactivation, and nucleotide‑binding oligomerization domain, leucine rich repeat, and pyrin domain‑containing protein 3 inflammasome inhibition. Nevertheless, recent findings show that SIRTs, specifically SIRT6, are also necessary for mounting an active inflammatory response in macrophages. SIRT6 has been shown to positively regulate tumor necrosis factor alpha (TNFα) secretion by demyristoylating pro-TNFα in the cytoplasm. However, how SIRT6, a nuclear chromatin-binding protein, fulfills this function in the cytoplasm is currently unknown. Herein, we show by Western blot and immunofluorescence that in macrophages and fibroblasts there is a subpopulation of SIRT6 that is highly unstable and quickly degraded via the proteasome. Upon lipopolysaccharide stimulation in Raw 264.7, bone marrow, and peritoneal macrophages, this population of SIRT6 is rapidly stabilized and localizes in the cytoplasm, specifically in the vicinity of the endoplasmic reticulum, promoting TNFα secretion. Furthermore, we also found that acute SIRT6 inhibition dampens TNFα secretion both in vitro and in vivo, decreasing lipopolysaccharide-induced septic shock. Finally, we tested SIRT6 relevance in systemic inflammation using an obesity-induced chronic inflammatory in vivo model, where TNFα plays a key role, and we show that short-term genetic deletion of SIRT6 in macrophages of obese mice ameliorated systemic inflammation and hyperglycemia, suggesting that SIRT6 plays an active role in inflammation-mediated glucose intolerance during obesity.     

GCAT|Panel,a comprehensive structural variant haplotype map of the Iberian population from high-coverage whole-genome sequencing

The combined analysis of haplotype panels with phenotype clinical cohorts is a common approach to explore the genetic architecture of human diseases. However, genetic studies are mainly based on single nucleotide variants (SNVs) and small insertions and deletions (indels). Here, we contribute to fill this gap by generating a dense haplotype map focused on the identification, characterization, and phasing of structural variants (SVs). By integrating multiple variant identification methods and Logistic Regression Models (LRMs), we present a catalogue of 35 431 441 variants, including 89 178 SVs (≥50 bp), 30 325 064 SNVs and 5 017 199 indels, across 785 Illumina high coverage (30x) whole-genomes from the Iberian GCAT Cohort, containing a median of 3.52M SNVs, 606 336 indels and 6393 SVs per individual. The haplotype panel is able to impute up to 14 360 728 SNVs/indels and 23 179 SVs, showing a 2.7-fold increase for SVs compared with available genetic variation panels. The value of this panel for SVs analysis is shown through an imputed rare Alu element located in a new locus associated with Mononeuritis of lower limb, a rare neuromuscular disease. This study represents the first deep characterization of genetic variation within the Iberian population and the first operational haplotype panel to systematically include the SVs into genome-wide genetic studies.     

Detection of altered protein conformations in living cells

The maturation, conformational stability, and the rate of in vivo degradation are specific for each protein and depend on both the intrinsic features of the protein and those of the surrounding cellular environment. While synthesis and degradation can be measured in living cells, stability and maturation of proteins are more difficult to quantify. We developed the split-ubiquitin method into a tool for detecting and analyzing changes in protein conformation. The biophysical parameter that forms the basis of these measurements is the time-averaged distance between the N terminus and C terminus of a protein. Starting from three proteins of known structure, we demonstrate the feasibility of this approach, and employ it to elucidate the effect of a previously described mutation in the protein Sec62p on its conformation in living cells.     

Hyperkalemic periodic paralysis M1592V mutation modifies activation in human skeletal muscle Na+ channel

Mutations in thehuman skeletal muscle Na⁺ channelunderlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na⁺ currents thought to depolarizethe sarcolemma and thus inactivate normalNa⁺ channels. We expressed humanwild-type or M₁₅₉₂V mutant-subunits with the ₁-subunitin Xenopus laevis oocytes and recordedNa⁺ currents using two-electrodeand cut-open oocyte voltage-clamp techniques. The most prominentfunctional difference betweenM₁₅₉₂V mutant and wild-typechannels is a 5- to 10-mV shift in the hyperpolarized direction of thesteady-state activation curve. The shift in the activation curve forthe mutant results in a larger overlap with the inactivation curve thanthat observed for wild-type channels. Accordingly, the current throughM₁₅₉₂V channels displays a larger noninactivating component than does that through wild-type channels atmembrane potentials near 40 mV. The functional properties of theM₁₅₉₂V mutant resemble those ofthe previously characterized HPPT₇₀₄M mutant. Both clinicallysimilar phenotypes arise from mutations located at a distance from theputative voltage sensor of the channel.

     

Selective decrease in paracellular conductance of tight junctions: role of the first extracellular domain of claudin-5

Claudin-5 is a protein component of many endothelial tight junctions, including those at the blood-brain barrier, a barrier that limits molecular exchanges between the central nervous system and the circulatory system. To test the contribution of claudin-5 to this barrier function of tight junctions, we expressed murine claudin-5 in Madin-Darby canine kidney II cells. The result was a fivefold increase in transepithelial resistance in claudin-5 transductants and a reduction in conductance of monovalent cations. However, the paracellular flux of neither neutral nor charged monosaccharides was significantly changed in claudin-5 transductants compared to controls. Therefore, expression of claudin-5 selectively decreased the permeability to ions. Additionally, site-directed mutations of particular amino acid residues in the first extracellular domain of claudin-5 altered the properties of the tight junctions formed in response to claudin-5 expression. In particular, the conserved cysteines were crucial: mutation of either cysteine abolishted the ability of claudin-5 to increase transepithelial resistance, and mutation of Cys(64) strikingly increased the paracellular flux of monosaccharides. These new insights into the functions of claudin-5 at the molecular level in tight junctions may account for some aspects of the blood-brain barrier's selective permeability.     

Angiopoietin decoy secreted at tumor site impairs tumor growth and metastases by inducing local inflammation and altering neoangiogenesis

The extracellular domain of the receptor tyrosine kinase Tie2/TEK (exTEK) has been used as an angiopoietin decoy to study the role of angiopoietins in the tumor–host interactions, using a syngeneic model of experimental metastases and subcutaneous tumor. Soluble exTEK secreted by transfected tumor cells inhibited HUVECs from forming tubes in Matrigel. ExTEK-transfected C26 colon carcinoma and TS/A mammary tumor cells displayed reduced growth rate when injected subcutaneously, and reduced ability to form experimental metastases when injected intravenously. Immunohistochemical analysis of tumors and metastases showed increased leukocytes infiltration and signs of inflammation in exTEK-secreting compared to parental tumor, as well as impairment in neo-vessel growth and organization. However, while neoangiogenesis eventually rescued in the subcutis, it failed to organize in the experimental metastases of exTEK-secreting tumor, contributing to the hampering of metastatic growth and to increased mice survival. The reactive infiltrate of C26TEK contained a different percentage of leukocytes and was responsible for the tumor inhibition. In fact, leukopenia induced by -irradiation of recipient mice or injection into interferon gamma (IFN-) gene knockout (GKO) mice resulted in reduced mouse survival and an increased number of lung metastases. On the other hand, interleukin (IL)-12 treatment prolonged the survival of mice bearing subcutaneous C26TEK but not of those bearing lung metastases, suggesting that IL-12 could exert further antiangiogenic effects at the site where the tumor can restore neoangiogenesis. These results show in vivo that reduced angiopoietin availability at the tumor site induces a local inflammatory response and impairment of neoangiogenesis which act synergistically to limit tumor growth and metastasis.Abbreviations AEC amino-ethylcarbazole - ELISA enzyme-linked immunosorbent assay - HRP horseradish peroxidase - HUVEC human umbilical vascular endothelial cell - i.v. intravenous - s.c. subcutaneous - TBS Tris-HCl buffered solution     

Differences in nocturnal basal and rhythmic prolactin secretion in untreated compared to treated HIV-infected men are associated with CD4+ T-lymphocytes

The existence of decreased hypothalamic dopaminergic tone in HIV-infected men has been suggested. In a cross-sectional study, we determined 12 h nocturnal basal and pulsatile prolactin (PRL) release levels (by blood sampling every 10 min) and their correlation with CD4+ T cells in seven volunteer HIV-negative, healthy men (group 1), and 21 normoprolactinemic, euthyroid, HIV-infected men divided into 3 groups (each group = 7): (i) group 2, asymptomatic HIV-infected stage A1 men, untreated; (ii) group 3, AIDS stage C3 without active opportunistic infections, untreated; and (iii) group 4, previously stage C3 after at least 6 months of successful highly active antiretroviral therapy. Serum PRL was measured by radioimmunoanalysis and the results were analysed by waveform-independent deconvolution analysis. CD4+ T lymphocytes were measured by flow cytometry and viral load by a nucleic acid sequence-based amplification assay. No differences were detected in the first two groups. In the third group, however, 100% of prolactin secretion was found to be pulsatile with a shorter secretory burst duration (P = 0.04), and a greater circulating half-life and pulse amplitude (P < or = 0.04). Group 4 had the greatest basal prolactin secretion (P < or = 0.04), and a shorter secretory burst duration (P = 0.04 vs group 2), circulating half-life (P = 0.01 vs group 3) and intersecretory burst interval (P = 0.06 vs group 1). PRL approximate entropy was similar among all groups. Linear correlations existed between CD4+ T cell counts and PRL secretory burst half duration (r = 0.62, P = 0.002) and amplitude (r = -0.63, P = 0.001), and in circulating serum half-life (r = - 0.61, P = 0.002) in HIV-infected groups. Viral load showed no correlations. It is suggested that differential changes in nocturnal prolactin secretion among HIV-infected men occurred while maintaining the normal coordinate feedback and/or feedforward control within the lactotropic axis. These changes may represent an adaptative mechanism to sustain, by different means, the maximal physiologic PRL production to stimulate the highest cellular immune response and/or reconstitution in attempting to survive.     

Intraoral lubrication of PRP-1, statherin and mucin as studied by AFM

The aim of this paper was to elucidate the mechanisms behind salivary lubrication with special emphasis on the lubricity of three key components of the pellicle, viz human acidic proline-rich protein 1 (PRP-1), human statherin and bovine submaxillary mucin (BSM). The lubricating properties of the proteins have been assessed by means of colloidal probe atomic force microscopy, and are discussed in relation to their adsorption behaviour. To various extents, the proteins investigated all showed a lubricating effect when adsorbed to silica surfaces. For comparable concentrations, PRP-1 was found to have a more pronounced lubricating effect than BSM, which in turn showed a higher lubricity than statherin. The relative lubricity is in accordance with previously reported relative adsorbed amounts of the three proteins, within the investigated concentration interval. It is concluded that PRP-1 has the highest lubricating capacity as a pure fraction among the preparations investigated, and that the lubricating effect of PRP-1 as a pure fraction is notably large as compared to the lubricity of human whole saliva.     

In situ assessment of porphyrin photosensitizers in Propionibacterium acnes

Porphyrins are known to be efficient photosensitizer molecules and the combined action of light and porphyrins in Propionibacterium acnes have a lethal action on the cells. Identification and quantification of in situ porphyrins in P. acnes have been done using an integrating sphere connected to an ordinary absorption spectrophotometer, and the amounts of porphyrins in the cells were quantified by measuring scattering free absorption spectra of the cell suspensions. The concentration of porphyrins in P. acnes cells were increased in either of two ways; by the addition of delta-aminolevulinic acid (ALA), which lead to the formation of coproporphyrin III under the incubation conditions used in these experiments, or by the addition of protoporphyrin IX (PPIX) to the cell suspension. In the latter case, PPIX molecules are taken up by the cells in a membrane-mediated uptake mechanism, and accumulate in the cells either on a monomeric or a particular aggregate form. The fraction of porphyrins on aggregate form increased with increasing PPIX additions. In the case of ALA induced porphyrin production, only monomeric porphyrins were stored in the cells. In both cases, the cells have a limited binding capacity of monomeric porphyrins, which is estimated to be 3 x 10(5) molecules/cell, or one porphyrin molecule to every 100st lipid molecule in the cell membrane.     

PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3(-/-) mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3(-/-) mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-alpha-trypsin inhibitor is normal in Ptx3(-/-) cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor alpha-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.