by
Elena Zaitseva;
Eugene Zaitsev;
Kamran Melikov;
Anush Arakelyan;
Mariana Marin;
Rafael Villasmil;
Leonid B. Margolis;
Gregory Melikian;
Leonid V. Chernomordik
HIV-1 entry into host cells starts with interactions between the viral envelope glycoprotein (Env) and cellular CD4 receptors and coreceptors. Previous work has suggested that efficient HIV entry also depends on intracellular signaling, but this remains controversial. Here we report that formation of the pre-fusion Env-CD4-coreceptor complexes triggers non-apoptotic cell surface exposure of the membrane lipid phosphatidylserine (PS). HIV-1-induced PS redistribution depends on Ca2+signaling triggered by Env-coreceptor interactions and involves the lipid scramblase TMEM16F. Externalized PS strongly promotes Env-mediated membrane fusion and HIV-1 infection. Blocking externalized PS or suppressing TMEM16F inhibited Env-mediated fusion. Exogenously added PS promoted fusion, with fusion dependence on PS being especially strong for cells with low surface density of coreceptors. These findings suggest that cell-surface PS acts as an important cofactor that promotes the fusogenic restructuring of pre-fusion complexes and likely focuses the infection on cells conducive to PS signaling. Zaitseva et al. show that HIV binding to target cells induces signaling that leads to exposure of phosphatidylserine on the cell surface. Interaction between the viral envelope glycoprotein and phosphatidylserine facilitates receptor-dependent merger of viral and cell membranes and infection. Phosphatidylserine dependence may focus infection on cells of certain activation status.
Tropomyosin, one of the major actin filament–binding proteins, regulates actin–myosin interaction and actin-filament stability. Multicellular organisms express a number of tropomyosin isoforms, but understanding of isoform-specific tropomyosin functions is incomplete. The nematode Caenorhabditis elegans has a single tropomyosin gene, lev-11, which has been reported to express four isoforms by using two separate promoters and alternative splicing. Here, we report a fifth tropomyosin isoform, LEV-11O, which is produced by alternative splicing that includes a newly identified seventh exon, exon 7a. By visualizing specific splicing events in vivo, we find that exon 7a is predominantly selected in a subset of the body wall muscles in the head, while exon 7b, which is the alternative to exon 7a, is utilized in the rest of the body. Point mutations in exon 7a and exon 7b cause resistance to levamisole-induced muscle contraction specifically in the head and the main body, respectively. Overex-pression of LEV-11O, but not LEV-11A, in the main body results in weak levamisole resistance. These results demonstrate that specific tropomyosin isoforms are expressed in the head and body regions of the muscles and contribute differentially to the regulation of muscle contractility.
Epigenetics allows for the inheritance of information in cellular lineages during differentiation, independent of changes to the underlying genetic sequence. This raises the question of whether epigenetic mechanisms also function in post-mitotic neurons. During the long life of the neuron, fluctuations in gene expression allow the cell to pass through stages of differentiation, modulate synaptic activity in response to environmental cues, and fortify the cell through age-related neuroprotective pathways. Emerging evidence suggests that epigenetic mechanisms such as DNA methylation and histone modification permit these dynamic changes in gene expression throughout the life of a neuron. Accordingly, recent studies have revealed the vital importance of epigenetic players in the central nervous system and during neurodegeneration. Here, we provide a review of several of these recent findings, highlighting novel functions for epigenetics in the fields of Rett syndrome, Fragile X syndrome, and Alzheimer's disease research. Together, these discoveries underscore the vital importance of epigenetics in human neurological disorders.
G quadruplex structures have been predicted by bioinformatics to form in the 5′- and 3′-untranslated regions (UTRs) of several thousand mature mRNAs and are believed to play a role in translation regulation. Elucidation of these roles has primarily been focused on the 3′-UTR, with limited focus on characterizing the G quadruplex structures and functions in the 5′-UTR. Investigation of the affinity and specificity of RNA binding proteins for 5′-UTR G quadruplexes and the resulting regulatory effects have also been limited. Among the mRNAs predicted to form a G quadruplex structure within the 5′-UTR is the survival motor neuron domain containing 1 (SMNDC1) mRNA, encoding a protein that is critical to the spliceosome. Additionally, this mRNA has been identified as a potential target of the fragile X mental retardation protein (FMRP), whose loss of expression leads to fragile X syndrome. FMRP is an RNA binding protein involved in translation regulation that has been shown to bind mRNA targets that form G quadruplex structures. In this study we have used biophysical methods to investigate G quadruplex formation in the 5′-UTR of SMNDC1 mRNA and analyzed its interactions with FMRP. Our results show that SMNDC1 mRNA 5′-UTR forms an intramolecular, parallel G quadruplex structure comprised of three G quartet planes, which is bound specifically by FMRP both in vitro and in mouse brain lysates. These findings suggest a model by which FMRP might regulate the translation of a subset of its mRNA targets by recognizing the G quadruplex structure present in their 5′-UTR, and affecting their accessibility by the protein synthesis machinery.
Interaction domains in Drosophila chromosomes form by segregation of active and inactive chromatin in the absence of CTCF loops, but the role of transcription versus other architectural proteins in chromatin organization is unclear. Here, we find that positioning of RNAPII via transcription elongation is essential in the formation of gene loops, which in turn interact to form compartmental domains. Inhibition of transcription elongation or depletion of cohesin decreases gene looping and formation of active compartmental domains. In contrast, depletion of condensin II, which also localizes to active chromatin, causes increased gene looping, formation of compartmental domains, and stronger intra-chromosomal compartmental interactions. Condensin II has a similar role in maintaining inter-chromosomal interactions responsible for pairing between homologous chromosomes, whereas inhibition of transcription elongation or cohesin depletion has little effect on homolog pairing. The results suggest distinct roles for cohesin and condensin II in the establishment of 3D nuclear organization in Drosophila.
Hypertonia is a neurological dysfunction associated with a number of central nervous system disorders, including cerebral palsy, Parkinson’s disease, dystonia, and epilepsy. Genetic studies have identified a homozygous truncation mutation in Trak1 that causes hypertonia in mice. Moreover, elevated Trak1 protein expression is associated with several types of cancers and variants in Trak1 are linked to childhood absence epilepsy in humans. Despite the importance of Trak1 in health and disease, the mechanisms of Trak1 action remain unclear and the pathogenic effects of Trak1 mutation are unknown. Here we report that Trak1 has a crucial function in regulation of mitochondrial fusion. Depletion of Trak1 inhibits mitochondrial fusion, resulting in mitochondrial fragmentation, whereas overexpression of Trak1 elongates and enlarges mitochondria. Our analyses revealed that Trak1 interacts and colocalizes with mitofusins on the outer mitochondrial membrane and functions with mitofusins to promote mitochondrial tethering and fusion. Furthermore, Trak1 is required for stress-induced mitochondrial hyperfusion and pro-survival response. We found that hypertonia-associated mutation impairs Trak1 mitochondrial localization and its ability to facilitate mitochondrial tethering and fusion. Our findings uncover a novel function of Trak1 as a regulator of mitochondrial fusion and provide evidence linking dysregulated mitochondrial dynamics to hypertonia pathogenesis.
Elevated sympathetic tone and activation of the renin-angiotensin system are pathophysiologic hallmarks of hypertension, and the interactions between these systems are particularly deleterious. The importance of Rho kinase as a mediator of the effects of angiotensin-II (AngII) in the periphery is clear, but the role of Rho kinase in sympathoexcitation caused by central AngII is not well established. We hypothesized that AngII mediates its effects in the brain by the activation of the RhoA/Rho kinase pathway. Chronically instrumented, conscious rabbits received the following intracerebroventricular infusion treatments for 2 weeks via osmotic minipump: AngII, Rho kinase inhibitor Fasudil, AngII plus Fasudil, or a vehicle control. AngII increased mean arterial pressure over the course of the infusion, and this effect was prevented by the coadministration of Fasudil. AngII increased cardiac and vascular sympathetic outflow as quantified by the heart rate response to metoprolol and the depressor effect of hexamethonium; coadministration of Fasudil abolished both of these effects. AngII increased baseline renal sympathetic nerve activity in conscious animals and impaired baroreflex control of sympathetic nerve activity; again Fasudil coinfusion prevented these effects. Each of these end points showed a statistically significant interaction between AngII and Fasudil. Quantitative immunofluorescence of brain slices confirmed that Rho kinase activity was increased by AngII and decreased by Fasudil. Taken together, these data indicate that hypertension, elevated sympathetic outflow, and baroreflex dysfunction caused by central AngII are mediated by Rho kinase activation and suggest that Rho kinase inhibition may be an important therapeutic target in sympathoexcitatory cardiovascular diseases.
by
Saurav Singh;
Alexander Grabner;
Christopher Yanucil;
Karla Schramm;
Brian Czaya;
Stefanie Krick;
Mark Czaja;
Rene Bartz;
Reimar Abraham;
Giovana Seno Di Marco;
Marcus Brand;
Myles Wolf;
Christian Faul
Patients with chronic kidney disease (CKD) develop increased levels of the phosphate-regulating hormone, fibroblast growth factor (FGF) 23, that are associated with a higher risk of mortality. Increases in inflammatory markers are another common feature that predicts poor clinical outcomes. Elevated FGF23 is associated with higher circulating levels of inflammatory cytokines in CKD, which can stimulate osteocyte production of FGF23. Here, we studied whether FGF23 can directly stimulate hepatic production of inflammatory cytokines in the absence of α-klotho, an FGF23 coreceptor in the kidney that is not expressed by hepatocytes. By a ctivating FGF receptor isoform 4 (FGFR4), FGF23 stimulated calcineurin signaling in cultured hepatocytes, which increased the expression and secretion of inflammatory cytokines, including C-reactive protein. Elevating serum FGF23 levels increased hepatic and circulating levels of C-reactive protein in wild-type mice, but not in FGFR4 knockout mice. Administration of an isoform-specific FGFR4 blocking antibody reduced hepatic and circulating levels of C-reactive protein in the 5/6 nephrectomy rat model of CKD. Thus, FGF23 can directly stimulate hepatic secretion of inflammatory cytokines. Our findings indicate a novel mechanism of chronic inflammation in patients with CKD and suggest that FGFR4 blockade might have therapeutic anti-inflammatory effects in CKD.
by
Elisabetta Manuela Foppiani;
Olivia Candini;
Ilenia Mastrolia;
Alba Murgia;
Giulia Grisendi;
Anna Valeria Samarelli;
Giulia Boscaini;
Lucrezia Pacchioni;
Massimo Pinelli;
Giorgio De Santis;
Edwin Horwitz;
Elena Veronesi;
Massimo Dominici
Background: The ex vivo expansion potential of mesenchymal stromal/stem cells (MSC) together with their differentiation and secretion properties makes these cells an attractive tool for transplantation and tissue engineering. Although the use of MSC is currently being tested in a growing number of clinical trials, it is still desirable to identify molecular markers that may help improve their performance both in vitro and after transplantation. Methods: Recently, HOXB7 was identified as a master player driving the proliferation and differentiation of bone marrow mesenchymal progenitors. In this study, we investigated the effect of HOXB7 overexpression on the ex vivo features of adipose mesenchymal progenitors (AD-MSC). Results: HOXB7 increased AD-MSC proliferation potential, reduced senescence, and improved chondrogenesis together with a significant increase of basic fibroblast growth factor (bFGF) secretion. Conclusion: While further investigations and in vivo models shall be applied for better understanding, these data suggest that modulation of HOXB7 may be a strategy for innovative tissue regeneration applications.
by
Chen Zhong;
Lahcen Jaafar;
Davies G Agyekum;
Haiyan Xiao;
Marlene F Wade;
R Ileng Kumaran;
David L Spector;
Gang Bao;
Matthew H Porteus;
William Dynan;
Steffen E Meiler
Engineered nucleases, which incise the genome at predetermined sites, have a number of laboratory and clinical applications. There is, however, a need for better methods for controlled intracellular delivery of nucleases. Here, we demonstrate a method for ligand-mediated delivery of zinc finger nucleases (ZFN) proteins using transferrin receptor-mediated endocytosis. Uptake is rapid and efficient in established mammalian cell lines and in primary cells, including mouse and human hematopoietic stem-progenitor cell populations. In contrast to cDNA expression, ZFN protein levels decline rapidly following internalization, affording better temporal control of nuclease activity. We show that transferrin-mediated ZFN uptake leads to site-specific in situ cleavage of the target locus. Additionally, despite the much shorter duration of ZFN activity, the efficiency of gene correction approaches that seen with cDNA-mediated expression. The approach is flexible and general, with the potential for extension to other targeting ligands and nuclease architectures.