Obesity-Linked PPARγ Ser273 Phosphorylation Promotes Beneficial Effects on the Liver, despite Reduced Insulin Sensitivity in Mice.

Since the removal of thiazolidinediones (TZDs) from the market, researchers have been exploring alternative anti-diabetic drugs that target PPARγ without causing adverse effects while promoting insulin sensitization by blocking serine 273 phosphorylation (Ser273 or S273). Nonetheless, the underlying mechanisms of the relationship between insulin resistance and S273 phosphorylation are still largely unknown, except for the involvement of growth differentiation factor (GDF3) regulation in the process. To further investigate potential pathways, we generated a whole organism knockin mouse line with a single S273A mutation (KI) that blocks the occurrence of its phosphorylation. Our observations of KI mice on different diets and feeding schedules revealed that they were hyperglycemic, hypoinsulinemic, presented more body fat at weaning, and presented an altered plasma and hepatic lipid profile, distinctive liver morphology and gene expression. These results suggest that total blockage of S273 phosphorylation may have unforeseen effects that, in addition to promoting insulin sensitivity, could lead to metabolic disturbances, particularly in the liver. Therefore, our findings demonstrate both the beneficial and detrimental effects of PPAR S273 phosphorylation and suggest selective modulation of this post translational modification is a viable strategy to treat type 2 diabetes.

Impaired expression of serine/arginine protein kinase 2 (SRPK2) affects melanoma progression.

Melanoma is one of the most aggressive tumors, and its lethality is associated with the ability of malignant cells to migrate and invade surrounding tissues to colonize distant organs and to generate widespread metastasis. The serine/arginine protein kinases 1 and 2 (SRPK1 and SRPK2) are classically related to the control of pre-mRNA splicing through SR protein phosphorylation and have been found overexpressed in many types of cancer, including melanoma. Previously, we have demonstrated that the pharmacological inhibition of SRPKs impairs pulmonary colonization of metastatic melanoma in mice. As the used compounds could target at least both SRPK1 and SRPK2, here we sought to obtain additional clues regarding the involvement of these paralogs in melanoma progression. We analyzed single-cell RNA sequencing data of melanoma patient cohorts and found that SRPK2 expression in melanoma cells is associated with poor prognosis. Consistently, CRISPR-Cas9 genome targeting of SRPK2, but not SRPK1, impaired actin polymerization dynamics as well as the proliferative and invasive capacity of B16F10 cells in vitro. In further in vivo experiments, genetic targeting of SRPK2, but not SRPK1, reduced tumor progression in both subcutaneous and caudal vein melanoma induction models. Taken together, these findings suggest different functional roles for SRPK1/2 in metastatic melanoma and highlight the relevance of pursuing selective pharmacological inhibitors of SRPK2.

Molecular and cellular basis of hyperassembly and protein aggregation driven by a rare pathogenic mutation in DDX3X.

Current studies estimate that 1-3% of females with unexplained intellectual disability (ID) present de novo splice site, nonsense, frameshift, or missense mutations in the DDX3X protein (DEAD-Box Helicase 3 X-Linked). However, the cellular and molecular mechanisms by which DDX3X mutations impair brain development are not fully comprehended. Here, we show that the ID-linked missense mutation L556S renders DDX3X prone to aggregation. By using a combination of biophysical assays and imaging approaches, we demonstrate that this mutant assembles solid-like condensates and amyloid-like fibrils. Although we observed greatly reduced expression of the mutant allele in a patient who exhibits skewed X inactivation, this appears to be enough to sequestrate healthy proteins into solid-like ectopic granules, compromising cell function. Therefore, our data suggest ID-linked DDX3X L556S mutation as a disorder arising from protein misfolding and aggregation.

Intracellular hyaluronic acid-binding protein 4 (HABP4): a candidate tumor suppressor in colorectal cancer.

Hyaluronic Acid-binding protein 4 (HABP4) is a regulatory protein of 57 kDa that is functionally involved in transcription regulation and RNA metabolism and shows several characteristics common to oncoproteins or tumor suppressors, including altered expression in cancer tissues, nucleus/cytoplasm shuttling, intrinsic lack of protein structure, complex interactomes and post translational modifications. Its gene has been found in a region on chromosome 9q22.3-31, which contains SNP haplotypes occurring in individuals with a high risk for familial colon cancer. To test a possible role of HABP4 in tumorigenesis we generated knockout mice by the CRISPR/Cas9 method and treated the animals with azoxymethane (AOM)/dextran sodium sulfate (DSS) for induction of colon tumors. HABP4-/- mice, compared to wild type mice, had more and larger tumors, and expressed more of the proliferation marker proteins Cyclin-D1, CDK4 and PCNA. Furthermore, the cells of the bottom of the colon crypts in the HABP4-/- mice divided more rapidly. Next, we generated also HABP4-/- HCT 116 cells, in cell culture and found again an increased proliferation in clonogenic assays in comparison to wild-type cells. Our study of the protein expression levels of HABP4 in human colon cancer samples, through immunohistochemistry assays, showed, that 30% of the tumors analyzed had low expression of HABP4. Our data suggest that HABP4 is involved in proliferation regulation of colon cells in vitro and in vivo and that it is a promising new candidate for a tumor suppressor protein that can be explored both in the diagnosis and possibly therapy of colon cancer.

MEF2C repressor variant deregulation leads to cell cycle re-entry and development of heart failure.

BACKGROUND: A pathophysiological link exists between dysregulation of MEF2C transcription factors and heart failure (HF), but the underlying mechanisms remain elusive. Alternative splicing of MEF2C exons α, ß and γ provides transcript diversity with gene activation or repression functionalities. METHODS: Neonatal and adult rat ventricular myocytes were used to overexpress MEF2C splicing variants γ+ (repressor) or γ-, or the inactive MEF2Cγ+23/24 (K23T/R24L). Phenotypic alterations in cardiomyocytes were determined by confocal and electron microscopy, flow cytometry and DNA microarray. We used transgenic mice with cardiac-specific overexpression of MEF2Cγ+ or MEF2Cγ- to explore the impact of MEF2C variants in cardiac phenotype. Samples of non-infarcted areas of the left ventricle from patients and mouse model of myocardial infarction were used to detect the expression of MEF2Cγ+ in failing hearts. FINDINGS: We demonstrate a previously unrealized upregulation of the transrepressor MEF2Cγ+ isoform in human and mouse failing hearts. We show that adenovirus-mediated overexpression of MEF2Cγ+ downregulates multiple MEF2-target genes, and drives incomplete cell-cycle reentry, partial dedifferentiation and apoptosis in the neonatal and adult rat. None of these changes was observed in cardiomyocytes overexpressing MEF2Cγ-. Transgenic mice overexpressing MEF2Cγ+, but not the MEF2Cγ-, developed dilated cardiomyopathy, correlated to cell-cycle reentry and apoptosis of cardiomyocytes. INTERPRETATION: Our results provide a mechanistic link between MEF2Cγ+ and deleterious abnormalities in cardiomyocytes, supporting the notion that splicing dysregulation in MEF2C towards the selection of the MEF2Cγ+ variant contributes to the pathogenesis of HF by promoting cardiomyocyte dropout. FUNDING: São Paulo Research Foundation (FAPESP); Brazilian National Research Council (CNPq).

Complex interactomes and post-translational modifications of the regulatory proteins HABP4 and SERBP1 suggest pleiotropic cellular functions.

The 57 kDa antigen recognized by the Ki-1 antibody, is also known as intracellular hyaluronic acid binding protein 4 and shares 40.7% identity and 67.4% similarity with serpin mRNA binding protein 1, which is also named CGI-55, or plasminogen activator inhibitor type-1-RNA binding protein-1, indicating that they might be paralog proteins, possibly with similar or redundant functions in human cells. Through the identification of their protein interactomes, both regulatory proteins have been functionally implicated in transcriptional regulation, mRNA metabolism, specifically RNA splicing, the regulation of mRNA stability, especially, in the context of the progesterone hormone response, and the DNA damage response. Both proteins also show a complex pattern of post-translational modifications, involving Ser/Thr phosphorylation, mainly through protein kinase C, arginine methylation and SUMOylation, suggesting that their functions and locations are highly regulated. Furthermore, they show a highly dynamic cellular localization pattern with localizations in both the cytoplasm and nucleus as well as punctuated localizations in both granular cytoplasmic protein bodies, upon stress, and nuclear splicing speckles. Several reports in the literature show altered expressions of both regulatory proteins in a series of cancers as well as mutations in their genes that may contribute to tumorigenesis. This review highlights important aspects of the structure, interactome, post-translational modifications, sub-cellular localization and function of both regulatory proteins and further discusses their possible functions and their potential as tumor markers in different cancer settings.

Opposing action of NCoR1 and PGC-1α in mitochondrial redox homeostasis.

The ability to respond to fluctuations of reactive oxygen species (ROS) within the cell is a central aspect of mammalian physiology. This dynamic process depends on the coordinated action of transcriptional factors to promote the expression of genes encoding for antioxidant enzymes. Here, we demonstrate that the transcriptional coregulators, PGC-1α and NCoR1, are essential mediators of mitochondrial redox homeostasis in skeletal muscle cells. Our findings reveal an antagonistic role of these coregulators in modulating mitochondrial antioxidant induction through Sod2 transcriptional control. Importantly, the activation of this mechanism by either PGC-1α overexpression or NCoR1 knockdown attenuates mitochondrial ROS levels and prevents cell death caused by lipid overload in skeletal muscle cells. The opposing actions of coactivators and corepressors, therefore, exert a commanding role over cellular antioxidant capacity.

Human Regulatory Protein Ki-1/57 Is a Target of SUMOylation and Affects PML Nuclear Body Formation.

Ki-1/57 is a nuclear and cytoplasmic regulatory protein first identified in malignant cells from Hodgkin's lymphoma. It is involved in gene expression regulation on both transcriptional and mRNA metabolism levels. Ki-1/57 belongs to the family of intrinsically unstructured proteins and undergoes phosphorylation by PKC and methylation by PRMT1. Previous characterization of its protein interaction profile by yeast two-hybrid screening showed that Ki-1/57 interacts with proteins of the SUMOylation machinery, the SUMO E2 conjugating enzyme UBC9 and the SUMO E3 ligase PIAS3, which suggested that Ki-1/57 could be involved with this process. Here we identified seven potential SUMO target sites (lysine residues) on Ki-1/57 sequence and observed that Ki-1/57 is modified by SUMO proteins in vitro and in vivo. We showed that SUMOylation of Ki-1/57 occurred on lysines 213, 276, and 336. In transfected cells expressing FLAG-Ki-1/57 wild-type, its paralog FLAG-CGI-55 wild-type, or their non-SUMOylated triple mutants, the number of PML-nuclear bodies (PML-NBs) is reduced compared with the control cells not expressing the constructs. More interestingly, after treating cells with arsenic trioxide (As2O3), the number of PML-NBs is no longer reduced when the non-SUMOylated triple mutant Ki-1/57 is expressed, suggesting that the SUMOylation of Ki-1/57 has a role in the control of As2O3-induced PML-NB formation. A proteome-wide analysis of Ki-1/57 partners in the presence of either SUMO-1 or SUMO-2 suggests that the involvement of Ki-1/57 with the regulation of gene expression is independent of the presence of either SUMO-1 or SUMO-2; however, the presence of SUMO-1 strongly influences the interaction of Ki-1/57 with proteins associated with cellular metabolism, maintenance, and cell cycle.

Defeating Bacterial Resistance and Preventing Mammalian Cells Toxicity Through Rational Design of Antibiotic-Functionalized Nanoparticles.

The rational synthesis of alternative materials is highly demanding due to the outbreak of infectious diseases and resistance to antibiotics. Herein, we report a tailored nanoantibiotic synthesis protocol where the antibiotic binding was optimized on the silver-silica core-shell nanoparticles surface to maximize biological responses. The obtained silver nanoparticles coated with mesoporous silica functionalized with ampicillin presented remarkable antimicrobial effects against susceptible and antibiotic-resistant Escherichia coli. In addition, these structures were not cell-death inducers and different steps of the mitotic cell cycle (prophase, anaphase and metaphase) were clearly identified. The superior biological results were attributed to a proper and tailored synthesis strategy.

Hydrocephalus and arthrogryposis in an immunocompetent mouse model of ZIKA teratogeny: A developmental study.

The teratogenic mechanisms triggered by ZIKV are still obscure due to the lack of a suitable animal model. Here we present a mouse model of developmental disruption induced by ZIKV hematogenic infection. The model utilizes immunocompetent animals from wild-type FVB/NJ and C57BL/6J strains, providing a better analogy to the human condition than approaches involving immunodeficient, genetically modified animals, or direct ZIKV injection into the brain. When injected via the jugular vein into the blood of pregnant females harboring conceptuses from early gastrulation to organogenesis stages, akin to the human second and fifth week of pregnancy, ZIKV infects maternal tissues, placentas and embryos/fetuses. Early exposure to ZIKV at developmental day 5 (second week in humans) produced complex manifestations of anterior and posterior dysraphia and hydrocephalus, as well as severe malformations and delayed development in 10.5 days post-coitum (dpc) embryos. Exposure to the virus at 7.5-9.5 dpc induces intra-amniotic hemorrhage, widespread edema, and vascular rarefaction, often prominent in the cephalic region. At these stages, most affected embryos/fetuses displayed gross malformations and/or intrauterine growth restriction (IUGR), rather than isolated microcephaly. Disrupted conceptuses failed to achieve normal developmental landmarks and died in utero. Importantly, this is the only model so far to display dysraphia and hydrocephalus, the harbinger of microcephaly in humans, as well as arthrogryposis, a set of abnormal joint postures observed in the human setting. Late exposure to ZIKV at 12.5 dpc failed to produce noticeable malformations. We have thus characterized a developmental window of opportunity for ZIKV-induced teratogenesis encompassing early gastrulation, neurulation and early organogenesis stages. This should not, however, be interpreted as evidence for any safe developmental windows for ZIKV exposure. Late developmental abnormalities correlated with damage to the placenta, particularly to the labyrinthine layer, suggesting that circulatory changes are integral to the altered phenotypes.

Ki-1/57 and CGI-55 ectopic expression impact cellular pathways involved in proliferation and stress response regulation.

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events.

Tailored silica-antibiotic nanoparticles: overcoming bacterial resistance with low cytotoxicity.

New and more aggressive antibiotic resistant bacteria arise at an alarming rate and represent an ever-growing challenge to global health care systems. Consequently, the development of new antimicrobial agents is required to overcome the inefficiency of conventional antibiotics and bypass treatment limitations related to these pathologies. In this study, we present a synthesis protocol, which was able to entrap tetracycline antibiotic into silica nanospheres. Bactericidal efficacy of these structures was tested against bacteria that were susceptible and resistant to antibiotics. For nonresistant bacteria, our composite had bactericidal efficiency comparable to that of free-tetracycline. On the other hand, the synthesized composites were able to avoid bacterial growth of resistant bacteria while free-tetracycline has shown no significant bactericidal effect. Finally, we have investigated the cytotoxicity of these nanoparticles against mammalian cells to check any possible poisoning effect. It was found that these nanospheres are not apoptosis-inducers and only a reduction on the cell replication rate was seen when compared to the control without nanoparticles.

The higher susceptibility of congenital analbuminemic rats to Ca2+-induced mitochondrial permeability transition is associated with the increased expression of cyclophilin D and nitrosothiol depletion.

Congenital analbuminemia is a rare autosomal recessive disorder characterized by a trace level of albumin in blood plasma and mild clinical symptoms. Analbuminemic patients generally present associated abnormalities, among which dyslipidemia is a hallmark. In this study, we show that mitochondria isolated from different tissues (liver, heart and brain) from 3-month-old analbuminemic rats (NAR) present a higher susceptibility to Ca(2+)-induced mitochondrial permeability transition (MPT), as assessed by either Ca(2+)-induced mitochondrial swelling, dissipation of membrane potential or mitochondrial Ca(2+) release. The Ca(2+) retention capacity of the liver mitochondria isolated from 3-month-old NAR was about 50% that of the control. Interestingly, the assessment of this variable in 21-day-old NAR indicated that the mitochondrial Ca(2+) retention capacity was preserved at this age, as compared to age-matched controls, which indicates that a reduced capacity for mitochondrial Ca(2+) retention is not a constitutive feature. The search for putative mediators of MPT sensitization in NAR revealed a 20% decrease in mitochondrial nitrosothiol content and a 30% increase in cyclophilin D expression. However, the evaluation of other variables related to mitochondrial redox status showed similar results between the controls and NAR, i.e., namely the contents of reduced mitochondrial membrane protein thiol groups and total glutathione, H(2)O(2) release rate, and NAD(P)H reduced state. We conclude that the higher expression of cyclophilin D, a major component of the MPT pore, and decreased nitrosothiol content in NAR mitochondria may underlie MPT sensitization in these animals.

Evidence for the association of the human regulatory protein Ki-1/57 with the translational machinery.

Ki-1/57 is a cytoplasmic and nuclear protein of 57 kDa first identified in malignant cells from Hodgkin's lymphoma. Based on yeast-two hybrid protein interaction we found out that Ki-1/57 interacts with adaptor protein RACK1 (receptor of activated kinase 1), CIRP (cold-inducible RNA-binding protein), RPL38 (ribosomal protein L38) and FXR1 (fragile X mental retardation-related protein 1). Since these proteins are involved in the regulation of translation we suspected that Ki-1/57 may have a role in it. We show by immunoprecipitation the association of Ki-1/57 with FMRP. Confocal microscopy revealed that Ki-1/57 colocalizes with FMRP/FXR1/2 to stress granules. Furthermore Ki-1/57 cosediments with free ribosomal particles and enhances translation, when tethered to a reporter mRNA, suggesting that Ki-1/57 may be involved in translational regulation.

Inhibitory effects of adenine nucleotides on brain mitochondrial permeability transition.

The adenine nucleotides ADP and ATP are probably the most important endogenous inhibitors of the mitochondrial permeability transition (MPT). We studied the inhibitory effects of adenine nucleotides on brain MPT by measuring mitochondrial swelling and Ca(2+) and cytochrome c release. We observed that in the presence of either ADP or ATP, at 250 µM, brain mitochondria accumulated more than 1 µmol Ca(2+) × mg protein(-1). ADP or ATP also prevented Ca(2+)-induced mitochondrial swelling and cytochrome c release. Interestingly, ATP lost most of its inhibitory effects on MPT when the experiments were carried out in the presence of ATP-regenerating systems. These results indicate that MPT inhibition observed in the presence of added ATP could be mainly due to hydrolysis of ATP to ADP. From mitochondrial swelling measurements, half-maximal inhibitory values (K(i)) of 4.5 and 98 µM were obtained for ADP and ATP, respectively. In addition, a delayed mitochondrial swelling sensitive to higher ADP concentrations was observed. Mitochondrial anoxia/reoxygenation did not interfere with the inhibitory effect of ADP on Ca(2+)-induced MPT, but oxidative phosphorylation markedly decreased this effect. We conclude that ADP is a potent inhibitor of brain MPT whereas ATP is a weaker inhibitor of this phenomenon. Our results suggest that ADP can have an important protective role against MPT-mediated tissue damage under conditions of brain ischemia and hypoglycemia.

3-nitropropionic acid-induced mitochondrial permeability transition: comparative study of mitochondria from different tissues and brain regions.

The adult rat striatum is particularly vulnerable to systemic administration of the succinate dehydrogenase inhibitor 3-nitropropionic acid (3NP), which is known to induce degeneration of the caudate-putamen, as occurs in Huntington's disease. The aim of the present study was to compare the susceptibility of isolated mitochondria from different rat brain regions (striatum, cortex, and cerebellum) as well as from the liver, kidney, and heart to mitochondrial permeability transition (MPT) induced by 3NP and Ca(2+). In the presence of micromolar Ca(2+) concentrations, 3NP induces MPT in a dose-dependent manner, as estimated by mitochondrial swelling and a decrease in the transmembrane electrical potential. A 3NP concentration capable of promoting a 10% inhibition of ADP-stimulated, succinate-supported respiration was sufficient to stimulate Ca(2+)-induced MPT. Brain and heart mitochondria were generally more sensitive to 3NP and Ca(2+)-induced MPT than mitochondria from liver and kidney. In addition, a partial inhibition of mitochondrial respiration by 3NP resulted in more pronounced MPT in striatal mitochondria than in cortical or cerebellar organelles. A similar inhibition of succinate dehydrogenase activity was observed in rat tissue homogenates obtained from various brain regions as well as from liver, kidney, and heart 24 hr after a single i.p. 3NP dose. Mitochondria isolated from forebrains of 3NP-treated rats were also more susceptible to Ca(2+)-induced MPT than those of control rats. We propose that the increased susceptibility of the striatum to 3NP-induced neurodegeneration may be partially explained by its susceptibility to MPT, together with the greater vulnerability of this brain region to glutamate receptor-mediated Ca(2+) influx.