PNAS Plus Significance Statements

Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis

Mark Hedglin, Binod Pandey, and Stephen J. Benkovic

The results from the reported studies indicate that the human lagging strand polymerase, pol δ, maintains a loose association with the sliding clamp, proliferating cell nuclear antigen (PCNA), while replicating and rapidly dissociates on stalling, leaving PCNA behind on the DNA. This behavior has profound implications on lagging strand synthesis as it limits the extent of processive DNA synthesis on undamaged DNA and promotes the rapid dissociation of pol δ on stalling at a replication-blocking lesion. This challenges the accepted models for polymerase recycling and exchange on the lagging strand and instead suggests passive mechanisms for the human system. These studies provide valuable insight for future experiments in the fields DNA replication, DNA repair, and DNA damage tolerance. (See pp. E1777–E1786.)

Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters

Jared T. Winkelman, Pete Chandrangsu, Wilma Ross, and Richard L. Gourse

Although it has long been appreciated that there are multiple RNA polymerase recognition elements in promoters, the determinants of transcription start site (TSS) selection are poorly understood. We find here that the absence of strong interactions between σ and the –10 element region of Escherichia coli rRNA promoters results in expansion of the transcription bubble prior to nucleotide addition, a phenomenon referred to as scrunching. Open complex scrunching accounts for the unusually long distance between the –10 element and the TSS and appears to contribute to the extraordinary activity of these promoters by minimizing abortive product formation, thereby increasing transcriptional output. These data provide insights into the determinants of TSS selection, promoter function, and promoter evolution. (See pp. E1787–E1795.)

Acetyl-CoA carboxylase inhibition by ND-630 reduces hepatic steatosis, improves insulin sensitivity, and modulates dyslipidemia in rats

Geraldine Harriman, Jeremy Greenwood, Sathesh Bhat, Xinyi Huang, Ruiying Wang, Debamita Paul, Liang Tong, Asish K. Saha, William F. Westlin, Rosana Kapeller, and H. James Harwood Jr.

Using structure-based drug design, we have identified a series of potent allosteric protein–protein interaction acetyl-CoA carboxylase inhibitors, exemplified by ND-630, that interact within the acetyl-CoA carboxylase subunit phosphopeptide acceptor and dimerization site to prevent dimerization and inhibit enzymatic activity. ND-630 reduces fatty acid synthesis and stimulates fatty acid oxidation in cultured cells and experimental animals, reduces hepatic steatosis, improves insulin sensitivity, reduces weight gain without affecting food intake, and favorably affects dyslipidemia in diet-induced obese rats and reduces hepatic steatosis, improves glucose-stimulated insulin secretion, and reduces hemoglobin A1c in Zucker diabetic fatty rats. These data suggest that ND-630 may be useful in treating a variety of metabolic disorders, including metabolic syndrome, type 2 diabetes, and fatty liver disease. (See pp. E1796–E1805.)

Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

Christian Molitor, Stephan Gerhard Mauracher, and Annette Rompel

Catechol oxidases and tyrosinases belong to the family of polyphenol oxidases (PPOs). In contrast to tyrosinases, catechol oxidases were so far defined to lack hydroxylase activity toward monophenols. Aurone synthase (AUS1) is a plant catechol oxidase that specializes in the conversion of chalcones to aurones (flower pigments). We evidence for the first time, to our knowledge, hydroxylase activity for a catechol oxidase (AUS1) toward its natural monophenolic substrate (chalcone). The presented first crystal structure of a plant pro-PPO provides insights into its activation mechanisms, and based on biochemical and structural studies of AUS1, we propose a novel catalytic reaction mechanism for plant PPOs. The proven hydroxylase functionality of AUS1 suggests that other catechol oxidases might also be involved in the plant’s secondary metabolism. (See pp. E1806–E1815.)

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

Sarah Schulz, Andreas Gietl, Katherine Smollett, Philip Tinnefeld, Finn Werner, and Dina Grohmann

DNA-dependent RNA polymerases (RNAPs) are complex enzymes that synthesize RNA in a factor-dependent fashion. Like mechanical engines, RNAPs consist of rigid and flexible parts; the catalytic function of RNAPs critically relies on conformational changes. Based on single-molecule FRET measurements that directly report on the movements of the RNAP clamp of the archaeal 12-subunit RNAP, we show that the clamp domain exists in alternative states distinguishable by the width of the DNA binding channel. The conformation of the clamp is adjusted through the transcription cycle; more precisely, it varies as a function of (i) RNA subunits Rpo4/7, (ii) the presence of the DNA nontemplate strand, and (iii) transcription initiation and elongation factors TFE and Spt4/5, respectively. (See pp. E1816–E1825.)

Identification of tissue-specific cell death using methylation patterns of circulating DNA

Roni Lehmann-Werman, Daniel Neiman, Hai Zemmour, Joshua Moss, Judith Magenheim, Adi Vaknin-Dembinsky, Sten Rubertsson, Bengt Nellgård, Kaj Blennow, Henrik Zetterberg, Kirsty Spalding, Michael J. Haller, Clive H. Wasserfall, Desmond A. Schatz, Carla J. Greenbaum, Craig Dorrell, Markus Grompe, Aviad Zick, Ayala Hubert, Myriam Maoz, Volker Fendrich, Detlef K. Bartsch, Talia Golan, Shmuel A. Ben Sasson, Gideon Zamir, Aharon Razin, Howard Cedar, A. M. James Shapiro, Benjamin Glaser, Ruth Shemer, and Yuval Dor

We describe a blood test for detection of cell death in specific tissues based on two principles: (i) dying cells release fragmented DNA to the circulation, and (ii) each cell type has a unique DNA methylation pattern. We have identified tissue-specific DNA methylation markers and developed a method for sensitive detection of these markers in plasma or serum. We demonstrate the utility of the method for identification of pancreatic β-cell death in type 1 diabetes, oligodendrocyte death in relapsing multiple sclerosis, brain cell death in patients after traumatic or ischemic brain damage, and exocrine pancreas cell death in pancreatic cancer or pancreatitis. The approach allows minimally invasive monitoring of tissue dynamics in humans in multiple physiological and pathological conditions. (See pp. E1826–E1834.)

Identifying genetic modulators of the connectivity between transcription factors and their transcriptional targets

Mina Fazlollahi, Ivor Muroff, Eunjee Lee, Helen C. Causton, and Harmen J. Bussemaker

We present a transcription-factor–centric computational method that utilizes data on natural variation in mRNA abundance to identify genetic loci that influence the responsiveness of genes to variation in the activities of their regulators. We call these connectivity quantitative trait loci, or cQTLs. When testing our method in yeast, we identified a polymorphism that modulates the mating response and confirmed our prediction experimentally. Our approach provides a sophisticated and nuanced approach to understanding the influence of genetic variation on interactions within regulatory networks. Applying our approach to human data may improve our understanding of the impact of genetic variation in contributing to phenotypic differences among individuals. (See pp. E1835–E1843.)

Force-producing ADP state of myosin bound to actin

Sarah F. Wulf, Virginie Ropars, Setsuko Fujita-Becker, Marco Oster, Goetz Hofhaus, Leonardo G. Trabuco, Olena Pylypenko, H. Lee Sweeney, Anne M. Houdusse, and Rasmus R. Schröder

The force-generating state of myosin bound to actin is a key state in the chemomechanical cycle of this motor, but the structural details of the force-generating state are unknown. CryoEM structures of myosin V bound to actin with and without nucleotide reveal a previously unidentified conformation of elements associated with nucleotide binding and, most importantly, of the β-sheet that controls the position of nucleotide-binding elements. These specific structural features explain how myosin can bind strongly to both actin and MgADP, allowing force to be developed. Thus, this structure provides fundamental insights into how myosin works. It also provides insights into how strain may prevent release of MgADP, which is a critical property for the cellular roles of many myosins, including myosin V. (See pp. E1844–E1852.)

Recognition of the disordered p53 transactivation domain by the transcriptional adapter zinc finger domains of CREB-binding protein

Alexander S. Krois, Josephine C. Ferreon, Maria A. Martinez-Yamout, H. Jane Dyson, and Peter E. Wright

The tumor suppressor p53 regulates the cellular response to genomic damage by recruiting the transcriptional coactivator cyclic-AMP response element-binding protein (CREB)-binding protein (CBP) and its paralog p300 to activate stress response genes. We report NMR structures of the complexes formed between the full-length, intrinsically disordered N-terminal transactivation domain of p53 and the transcriptional adapter zinc finger domains (TAZ1 and TAZ2) of CBP. Exchange broadening of NMR spectra of the complexes was ameliorated by using fusion proteins and segmental isotope labeling. The structures show how the p53 transactivation domain uses bipartite binding motifs to recognize diverse partners, reveal the critical interactions required for high affinity binding, and provide insights into the mechanism by which phosphorylation enhances the ability of p53 to recruit CBP and p300. (See pp. E1853–E1862.)

A RhoA and Rnd3 cycle regulates actin reassembly during membrane blebbing

Kana Aoki, Fumiyo Maeda, Tomoya Nagasako, Yuki Mochizuki, Seiichi Uchida, and Junichi Ikenouchi

The plasma membrane and the underlying actin cortex show dynamic interactions. When the plasma membrane detaches from the actin cortex, the plasma membrane protrudes. The protruded membrane is called a membrane bleb and is often observed during cell migration or cytokinesis. In the present study, we determined the molecular mechanisms involved in the reassembly of the actin cortex in membrane blebs using live-cell imaging. (See pp. E1863–E1871.)

Mitochondrial calcium uniporter regulator 1 (MCUR1) regulates the calcium threshold for the mitochondrial permeability transition

Dipayan Chaudhuri, Daniel J. Artiga, Sunday A. Abiria, and David E. Clapham

Cells injured by a variety of stressors feature a form of mitochondrial dysfunction termed the permeability transition. During this process, mitochondria swell and become disrupted, ultimately leading to cell death. In excitable cells such as cardiomyocytes or neurons, such injury is often triggered by calcium overload. By screening Drosophila cells, we have found a protein, mitochondrial calcium uniporter regulator 1 (MCUR1), that appears to regulate the amount of calcium required to induce the permeability transition. Modulating the function of this protein acutely may prove beneficial in limiting tissue damage during diseases that feature calcium overload. (See pp. E1872–E1880.)

Re-evaluation of the roles of DROSHA, Exportin 5, and DICER in microRNA biogenesis

Young-Kook Kim, Boseon Kim, and V. Narry Kim

MicroRNAs (miRNAs) are noncoding RNAs with diverse roles in development and pathogenesis. Biogenesis of canonical miRNA requires nuclear processing by DROSHA, nuclear export by Exportin 5, and cytoplasmic processing by DICER. To gain a deeper understanding of the maturation processes, we here ablated the DROSHA, Exportin 5, and DICER genes using the same human cell line. Canonical miRNA production was abolished in DROSHA-deleted cells, revealing an irreplaceable role of DROSHA. Interestingly, however, some canonical miRNAs were still produced without DICER albeit at markedly reduced levels, and many were detected in Exportin 5-deleted cells at only modestly decreased levels. Our study allows us to understand differential contributions of key biogenesis factors, and provides valuable resources for miRNA research. (See pp. E1881–E1889.)

Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer

Jun Huang, Xun Zeng, Natalia Sigal, Peder J. Lund, Laura F. Su, Huang Huang, Yueh-hsiu Chien, and Mark M. Davis

The recognition of foreign peptide-MHCs by T cells is a central event in adaptive immunity that triggers antigen-specific immune responses against infections and cancer. To study antigen-specific T cells, we devised a peptide-MHC dodecamer that can sensitively detect and specifically stain these T cells, especially low-affinity and rare ones. This dodecamer technology is superior to most current peptide-MHC multimers, compatible with existing reagents, inexpensive to make, and easy to use. It has been successfully applied to studies of human and murine antigen-specific αβ and γδ T cells by flow cytometry and mass cytometry. Thus, this dodecamer constitutes an important tool for the investigation of antigen-specific T cells in basic and clinical research. (See pp. E1890–E1897.)

Soluble (pro)renin receptor via β-catenin enhances urine concentration capability as a target of liver X receptor

Xiaohan Lu, Fei Wang, Chuanming Xu, Sunny Soodvilai, Kexin Peng, Jiahui Su, Long Zhao, Kevin T. Yang, Yumei Feng, Shu-Feng Zhou, Jan-Åke Gustafsson, and Tianxin Yang

The soluble (pro)renin receptor (sPRR) is produced by protease-mediated cleavage of PRR and is elevated under certain pathological conditions. To our knowledge, no prior studies have reported the biological function of sPRR in general or the antidiuretic function of the soluble protein in particular. Here we describe a previously unreported role of sPRR in the enhancement of renal aquaporin 2 (AQP2) expression and urine-concentrating capability. We further show that sPRR acts via frizzled class receptor 8-depdendent β-catenin signaling to increase AQP2 expression in the collecting duct cells. These findings offer an unreported insight into the physiological role of sPRR in regulating fluid homeostasis. In addition, we found that liver X receptor activation by TO901317 resulted in diabetes insipidus because of the inhibition of renal PRR expression. (See pp. E1898–E1906.)

Viral serine palmitoyltransferase induces metabolic switch in sphingolipid biosynthesis and is required for infection of a marine alga

Carmit Ziv, Sergey Malitsky, Alaa Othman, Shifra Ben-Dor, Yu Wei, Shuning Zheng, Asaph Aharoni, Thorsten Hornemann, and Assaf Vardi

This work investigates the metabolic basis of the interactions between the cosmopolitan bloom-forming alga Emiliania huxleyi and its specific large DNA virus. We demonstrate some of the basic metabolic principles used by the EhV virus to “engineer” its host sphingolipid metabolism, to produce a unique suite of virus-specific lipids. These sphingolipids were essential for the virus assembly and infectivity. These results present novel insight into the chemical “arm race” at sea that coevolved around a unique metabolic pathway. Furthermore, it may provide important evolutionary and biochemical insights into sphingolipid biosynthesis and its functional role in other host–pathogen interactions (e.g. HIV, hepatitis C virus, dengue virus). (See pp. E1907–E1916.)

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

Morgan Beeby, Deborah A. Ribardo, Caitlin A. Brennan, Edward G. Ruby, Grant J. Jensen, and David R. Hendrixson

Many bacteria swim using helical propellers, flagella. Intriguingly, different bacteria show different swimming abilities, strikingly illustrated by the abilities of some to bore through viscous fluids (e.g., gastrointestinal mucus) in which others are completely immobilized. We used 3D electron microscopy to show that differences can be explained by the structures of the torque-generating motors: two diverse high-torque motors position additional torque-generating complexes at wider radii from the axial driveshaft than in the model enteric bacteria; this positioning is consistent with the exertion of greater leverage to rotate the flagellum and thus greater torque generation. Intriguingly, these torque-generating complexes are scaffolded at wider radii by a conserved but divergent family of structures, suggesting an ancient origin of reconfiguring torque output. (See pp. E1917–E1926.)

Phasic, suprathreshold excitation and sustained inhibition underlie neuronal selectivity for short-duration sounds

Rishi K. Alluri, Gary J. Rose, Jessica L. Hanson, Christopher J. Leary, Gustavo A. Vasquez-Opazo, Jalina A. Graham, and Jeremy Wilkerson

Understanding how excitatory and inhibitory inputs are integrated to achieve sensory selectivity is an important, but elusive, goal in neuroscience. Whole-cell recordings in vivo have revealed excitation and inhibition, but the computational mechanisms are generally unclear. In the inferior colliculus, neurons exist that respond selectively to short-duration sounds; the mechanistic basis of this selectivity has long been debated. We combined whole-cell recordings, in vivo, with conductance extraction algorithms and focal pharmacological manipulations to show that duration selectivity results from an anticoincidence of suprathreshold excitation and inhibition. These results challenge the widely held view that postinhibitory rebound depolarization must coincide with subthreshold excitation to generate duration selectivity. (See pp. E1927–E1935.)

Four-dimensional maps of the human somatosensory system

Pietro Avanzini, Rouhollah O. Abdollahi, Ivana Sartori, Fausto Caruana, Veronica Pelliccia, Giuseppe Casaceli, Roberto Mai, Giorgio Lo Russo, Giacomo Rizzolatti, and Guy A. Orban

Here, we show how anatomical and functional data recorded from patients undergoing stereo-EEG can be combined to generate highly resolved four-dimensional maps of human cortical processing. We used this technique, which provides spatial maps of the active cortical nodes at a millisecond scale, to depict the somatosensory processing following electrical stimulation of the median nerve in nearly 100 patients. The results showed that human somatosensory system encompasses a widespread cortical network including a phasic component, centered on primary somatosensory cortex and neighboring motor, premotor, and inferior parietal regions, as well as a tonic component, centered on the opercular and insular areas, lasting more than 200 ms. (See pp. E1936–E1943.)

Gene deficiency and pharmacological inhibition of soluble epoxide hydrolase confers resilience to repeated social defeat stress

Qian Ren, Min Ma, Tamaki Ishima, Christophe Morisseau, Jun Yang, Karen M. Wagner, Ji-chun Zhang, Chun Yang, Wei Yao, Chao Dong, Mei Han, Bruce D. Hammock, and Kenji Hashimoto

Depression is the most common and debilitating psychiatric disorder in the world. However, the precise mechanisms underlying depression remain largely unknown. Recent evidence suggests that soluble epoxide hydrolase (sEH) plays a key role in inflammation, which is involved in depression. The sEH inhibitor, TPPU, showed antidepressant effects in animal models of depression. Expression of sEH protein was increased in the brain of chronically stressed (susceptible) mice and depressed patients. Prophylactic sEH inhibition or sEH-KO resulted in resilience to repeated social defeat stress, associated with increased BDNF-TrkB signaling in prefrontal cortex and hippocampus of KO mice. This study shows that sEH plays a key role in the pathophysiology of depression, and that its inhibitors could be potential therapeutic drugs for depression. (See pp. E1944–E1952.)

Biphasic regulation of InsP₃ receptor gating by dual Ca²⁺ release channel BH3-like domains mediates Bcl-x_L control of cell viability

Jun Yang, Horia Vais, Wenen Gu, and J. Kevin Foskett

Changes in Ca²⁺ concentration in the cell play important roles in cell life and death decisions. Antiapoptotic Bcl-2 family proteins help protect cells from dying by interacting with proteins at mitochondria and endoplasmic reticulum. At the endoplasmic reticulum, antiapoptotic Bcl-2 proteins interact with InsP₃R Ca²⁺ channels that release Ca²⁺ into the cytoplasm. However, it is controversial how they interact with the InsP₃R, as well as the functional consequences of the interactions. We found that antiapoptotic Bcl-x_L interacts with InsP₃Rs by unique mechanisms that change the activity of the channel depending on its concentration. We also found that disrupting these interactions diminishes cell viability. Our results provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP₃R. (See pp. E1953–E1962.)