by
Laura Bryant;
Dong Li;
Samuel G. Cox;
Dylan Marchione;
Evan F. Joiner;
Khadija Wilson;
Kevin Janssen;
Pearl Lee;
Michael E. March;
Divya Nair;
Elliott Sherr;
Brieana Fregeau;
Klaas J. Wierenga;
Alexandrea Wadley;
Grazia M. S. Mancini;
Nina Powell-Hamilton;
Jiddeke van de Kamp;
Theresa Grebe;
Juanita Neira;
Elizabeth J. Bhoj
Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation.
Low complexity protein sequences are often intrinsically unstructured and many have the potential to polymerize into amyloid aggregates including filaments and hydrogels. RNA-binding proteins are unusually enriched in such sequences raising the question as to what function these domains serve in RNA metabolism. One such yeast protein, Nab3, is an 802 amino acid termination factor that contains an RNA recognition motif and a glutamine/proline rich domain adjacent to a region with structural similarity to a human hnRNP. A portion of the C-terminal glutamine/proline-rich domain assembles into filaments that organize into a hydrogel. Here we analyze the determinants of filament formation of the isolated low complexity domain as well as examine the polymerization properties of full-length Nab3. We found that the C-terminal region with structural homology to hnRNP-C is not required for assembly, nor is an adjacent stretch of 16 glutamines. However, reducing the overall glutamine composition of this 134-amino acid segment from 32% to 14% destroys its polymerization ability. Importantly, full-length wildtype Nab3 also formed filaments with a characteristic cross-β structure which was dependent upon the glutamine/proline-rich region. When full length Nab3 with reduced glutamine content in its low complexity domain was exchanged for wildtype Nab3, cells were not viable. This suggests that polymerization of Nab3 is normally required for its function. In an extension of this idea, we show that the low complexity domain of another yeast termination factor, Pcf11, polymerizes into amyloid fibers and a hydrogel. These findings suggest that, like many other RNA binding proteins, termination factors share a common biophysical trait that may be important for their function.
Arif et al. report that phosphorylation of the important metabolism-controlling kinase, S6K1, at two sites near the protein terminus induces its phosphorylation of multiple targets related to lipid metabolism. These insulin-stimulated phosphorylation events in adipocytes (fat cells) might contribute to the known influence of S6K1 on obesity.
Overcoming therapeutic resistance in glioblastoma (GBM) is an essential strategy for improving cancer therapy. However, cancer cells possess various evasion mechanisms, such as metabolic reprogramming, which promote cell survival and limit therapy. The diverse metabolic fuel sources that are produced by autophagy provide tumors with metabolic plasticity and are known to induce drug or radioresistance in GBM. This study determined that autophagy, a common representative cell homeostasis mechanism, was upregulated upon treatment of GBM cells with ionizing radiation (IR). Nuclear receptor binding factor 2 (NRBF2)—a positive regulator of the autophagy initiation step—was found to be upregulated in a GBM orthotopic xenograft mouse model. Furthermore, ATP production and the oxygen consumption rate (OCR) increased upon activation of NRBF2-mediated autophagy. It was also discovered that changes in metabolic state were induced by alterations in metabolite levels caused by autophagy, thereby causing radioresistance. In addition, we found that lidoflazine—a vasodilator agent discovered through drug repositioning—significantly suppressed IR-induced migration, invasion, and proliferation by inhibiting NRBF2, resulting in a reduction in autophagic flux in both in vitro models and in vivo orthotopic xenograft mouse models. In summary, we propose that the upregulation of NRBF2 levels reprograms the metabolic state of GBM cells by activating autophagy, thus establishing NRBF2 as a potential therapeutic target for regulating radioresistance of GBM during radiotherapy.
by
Qiang Wen;
Benjamin Goldenson;
Serena J. Silver;
Monica Schenone;
Vladimir Dancik;
Zan Huang;
Ling-Zhi Wang;
Timothy Lewis;
W. Frank An;
Xiaoyu Li;
Mark-Anthony Bray;
Clarisse Thiollier;
Lauren Diebold;
Laure Gilles;
Martha S. Vokes;
Christopher B. Moore;
Meghan Bliss-Moreau;
Lynn VerPlank;
Nicola J. Tolliday;
William G Woods
The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.
Ca2+-activated Cl− channels (CaCCs) perform a multitude of functions including the control of cell excitability, regulation of cell volume and ionic homeostasis, exocrine and endocrine secretion, fertilization, amplification of olfactory sensory function, and control of smooth muscle cell contractility. CaCCs are the translated products of two members (ANO1 and ANO2, also known as TMEM16A and TMEM16B) of the Anoctamin family of genes comprising ten paralogs. This review focuses on recent progress in understanding the molecular mechanisms involved in the regulation of ANO1 by cytoplasmic Ca2+, post-translational modifications, and how the channel protein interacts with membrane lipids and protein partners. After first reviewing the basic properties of native CaCCs, we then present a brief historical perspective highlighting controversies about their molecular identity in native cells. This is followed by a summary of the fundamental biophysical and structural properties of ANO1. We specifically address whether the channel is directly activated by internal Ca2+ or indirectly through the intervention of the Ca2+-binding protein Calmodulin (CaM), and the structural domains responsible for Ca2+- and voltage-dependent gating. We then review the regulation of ANO1 by internal ATP, Calmodulin-dependent protein kinase II-(CaMKII)-mediated phosphorylation and phosphatase activity, membrane lipids such as the phospholipid phosphatidyl-(4,5)-bisphosphate (PIP2), free fatty acids and cholesterol, and the cytoskeleton. The article ends with a survey of physical and functional interactions of ANO1 with other membrane proteins such as CLCA1/2, inositol trisphosphate and ryanodine receptors in the endoplasmic reticulum, several members of the TRP channel family, and the ancillary Κ+ channel β subunits KCNE1/5.
by
Daniel DiToro;
Colleen J. Winstead;
Pharm Duy;
Steven Witte;
Rakieb Andargachew;
Jeffrey R. Singer;
C. Garrett Wilson;
Carlene L. Zindl;
Rita J. Luther;
Daniel J. Silberger;
Benjamin T. Weaver;
E. Motunrayo Kolawole;
Ryan J. Martinez;
Henriatta Turner;
Robin D. Hatton;
James J. Moon;
Sing Sing Way;
Brian Evavold;
Casey T. Weaver
RNA sequencing of naïve T cells sorted on the basis of IL-2 reporter expression identified cosegregation of transcripts encoding IL-2 and Bcl6-the signature transcription factor of TFH cells. Conversely, IL-2-negative (IL-2-) cells preferentially expressed the gene Prdm1, which encodes the transcriptional repressor Blimp1. Blimp1, in turn, antagonizes Bcl6 and the TFH developmental program. This suggested that IL-2 producers give rise to TFH cells, whereas IL-2 nonproducers give rise to non-TFH effector cells. Moreover, the fact that IL-2 receptor signaling induces expression of Prdm1 via Stat5 suggested that IL-2 producers resisted IL-2 signaling and activated IL-2 signaling in nonproducers in trans. Indeed, in vivo studies established that IL-2 signaling was mostly paracrine and that depletion of IL-2- producing cells selectively impaired TFH cell development. Finally, IL-2 expression was limited to a subset of naïve T cells that received the strongest T cell receptor (TCR) signals, establishing a link between TCR signal strength, IL-2 production, and TFH versus non-TFH differentiation. This study provides newinsights into themechanisms that control early bifurcation of CD4+ T cells into TFH and non-TFH effectors. Naïve T cells that receive differing strengths of TCR signals stratify into those that exceed a threshold predisposing them to IL-2 production and early TFH commitment and those that do not express IL-2 yet receive IL-2 signaling, which reinforces non-TFH effector commitment.
Sepsis, a pathology resulting from excessive inflammatory response that leads to multiple organ failure, is a major cause of mortality in intensive care units. Macrophages play an important role in the pathophysiology of sepsis. Accumulating evidence has suggested an upregulated rate of aerobic glycolysis as a key common feature of activated proinflammatory macrophages. Here, we identified a crucial role of myeloid 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (Pfkfb3), a glycolytic activator in lipopolysaccharide (LPS)-induced endotoxemia in mice. Pfkfb3 expression is substantially increased in bone marrow derived macrophages (BMDMs) treated with LPS in vitro and in lung macrophages of mice challenged with LPS in vivo. Myeloid-specific knockout of Pfkfb3 in mice protects against LPS-induced lung edema, cardiac dysfunction and hypotension, which were associated with decreased expression of interleukin 1 beta (Il1b), interleukin 6 (Il6) and nitric oxide synthase 2 (Nos2), as well as reduced infiltration of neutrophils and macrophages in lung tissue. Pfkfb3 ablation in cultured macrophages attenuated LPS-induced glycolytic flux, resulting in a decrease in proinflammatory gene expression. Mechanistically, Pfkfb3 ablation or inhibition with a Pfkfb3 inhibitor AZ26 suppresses LPS-induced proinflammatory gene expression via the NF-κB signaling pathway. In summary, our study reveals the critical role of Pfkfb3 in LPS-induced sepsis via reprogramming macrophage metabolism and regulating proinflammatory gene expression. Therefore, PFKFB3 is a potential target for the prevention and treatment of inflammatory diseases such as sepsis.
Ras-related C3 botulinum toxin substrate 1 (Rac1) is a small GTPase that is well known for its sensitivity to the environmental stress of a cell or an organism. It senses the external signals which are transmitted from membrane-bound receptors and induces downstream signaling cascades to exert its physiological functions. Rac1 is an important regulator of a variety of cellular processes, such as cytoskeletal organization, generation of oxidative products, and gene expression. In particular, Rac1 has a significant influence on certain brain functions like neuronal migration, synaptic plasticity, and memory formation via regulation of actin dynamics in neurons. Abnormal Rac1 expression and activity have been observed in multiple neurological diseases. Here, we review recent findings to delineate the role of Rac1 signaling in neurodevelopmental disorders associated with abnormal spine morphology, synaptogenesis, and synaptic plasticity. Moreover, certain novel inhibitors of Rac1 and related pathways are discussed as potential avenues toward future treatment for these diseases.