bluuebunny/crossref_metadata_2025_split huggingface dataset:
Are Fibrillin‐Notch Interactions Important in Development and Disease?
Authors: Sakai Lynn Y. , Carlson Eric J. 
Published: April 2020

In humans, mutations in the gene for fibrillin‐1 (FBN1) cause both the Marfan syndrome as well as several types of acromelic dysplasias. Musculoskeletal features of the Marfan syndrome (long bone overgrowth and arachnodactyly, joint hypermobility, and a muscle wasting phenotype) are the opposite of those in acromelic dysplasias (short stature and brachydactyly, joint stiffness, and a hypermuscular build). How mutations in the same gene can result in opposite phenotypes is poorly understood. Mutations in FBN2 cause congenital contractural arachnodactyly, which features a mixture of related musculoskeletal features (muscle hypoplasia, contractures of the large and small joints and arachnodactyly).The fibrillins are multifunctional glycoproteins that polymerize into extracellular matrix microfibrils. Although we have associated fibrillin microfibril structure with the opposite musculoskeletal features of Marfan syndrome and acromelic dysplasias, we can only speculate that perturbations in growth factor signaling are involved. To begin to address this gap in our understanding, we hypothesize that direct interactions between fibrillin and cells coordinate growth factor signaling. Therefore, we searched for interactions between fibrillin and cellular receptors.Fibrillins, like Notch receptors and Notch ligands, are composed of multiple tandemly repeated cbEGF‐like domains. Each fibrillin contains 43 cbEGF‐like domains. What if Notch receptors or ligands also bind to fibrillins in the extracellular matrix? Our biochemical data imply that fibrillin interactions with Notch receptors or ligands are just as likely to occur in vivo as interactions between Notch receptors and known Notch transmembrane ligands. Human genetic evidence support significant roles for Notch signaling components in vascular and musculoskeletal diseases, tissues primarily affected in the fibrillinopathies.The current concept of Notch signaling is based strictly on cell‐cell communication. Our investigations have the potential to transform current understanding of context‐dependent Notch signaling by introducing the possibility that Notch signaling components also interact with fibrillin extracellular matrix.Support or Funding Informationgrants from the Shriners Hospitals for Children to Lynn Y. Sakai
Chromatin Regulation and Genome Maintenance by Mammalian SIRT6 and SIRT7
Authors: Chua Katrin F. 
Published: April 2020

Members of the Sirtuin family of enzymes are important regulators of genomic stability, stress responses, and metabolic programs that impact on human physiology, aging, and age‐related disease processes. We previously showed that the mammalian Sirtuins SIRT6 and SIRT7 have high‐selectivity histone deacetylase activities at chromatin, and inactivation of SIRT6 or SIRT7 results in dysregulated histone acetylation states and gene expression programs, with pathological consequences at the cellular and whole organism levels. Recently, we have been exploring novel functions of SIRT6 and SIRT7 in silencing of heterochromatic regions of the genome, the deregulation of which has been linked to aging and cancer biology. We found that pericentric heterochromatin silencing by SIRT6 prevents acute cellular senescence that is triggered by pathologic pericentric transcripts. We also uncovered a second novel trigger of human cellular senescence, ribosomal DNA instability in nucleoli, and we showed that SIRT7 guards against senescence induced by this instability. In our studies, a long‐term focus has been identifying substrates of SIRT6 and SIRT7 and their roles in aging and disease pathways. In new work, we have identified a novel physiologic substrate of SIRT7 at chromatin, and we are characterizing the genomic landscape of this SIRT7‐dependent deacetylation target, and its downstream chromatin and nuclear signaling mechanisms. We will also discuss mechanistic insights into the functions of SIRT6 and SIRT7 from new proteomic, cellular, and mouse model studies.Support or Funding InformationVA Merit Review NIH/NIA
tRNA: Splicing and Intron Turnover
Authors: Hopper Anita K. , Nostramo Regina T. , Bao Alicia , Peltier Lauren M. 
Published: April 2020

In most eukaryotic genomes a subset of tRNA genes contains introns. Introns in tRNAs genes are short sequences located 1 nt. 3′ of the anticodon. Introns are removed from precursor tRNAs (pre‐tRNA) by a conserved heterotetrameric protein enzyme, tRNA splicing endonuclease (SEN), that recognizes pre‐tRNA structure rather than sequence motifs at splice junctions. In vertebrate cells intron removal from pre‐tRNAs occurs in the nucleoplasm; in contrast, pre‐tRNA splicing in budding yeast was surprisingly discovered to occur on the mitochondrial surface (Yoshihisa et al. 2003). We recently reported that the SEN complex also locates to the mitochondrial surface in fission yeast (Wan & Hopper 2018) and, therefore, pre‐tRNA splicing on the mitochondrial surface has been conserved for at least 500 million years. The mitochondrial outer membrane protein, Tom70, is required for efficient SEN subunit location to mitochondria in both budding and fission yeast (Wan & Hopper 2018). The freed introns are rarely detected in cells and until recently it was unknown how are they are turned over. We identified at least 5 separate, species‐specific mechanisms that function in tRNA intron destruction (Wu & Hopper, 2014; Bao, unpublished). Interestingly, under particular stress conditions, specific tRNA introns accumulate, possibly indicating their functions in stress response (Peltier, unpublished). Moreover, we discovered that tRNA introns possess long stretches of perfect complementarity to particular budding yeast mRNAs (Bao, unpublished), also indicative of their possible roles in gene regulation. Our initial experiments support the hypothesis that tRNA introns may function as a novel type of noncoding regulatory RNA (Nostramo, unpublished).Support or Funding InformationNIH GMS 122884
Wiring the powerhouse: Systems‐to‐structure approaches for defining mitochondrial protein function
Authors: Pagliarini David 
Published: April 2020

Mitochondria—epicenters of cellular metabolism and bioenergetics—are much less well understood than their iconic “powerhouse” moniker implies. Hundreds of mitochondrial proteins remain uncharacterized, classic pathways include many steps not yet assigned to specific enzyme functions, a large percentage of mitochondrial diseases remain unresolved, and there are no approved FDA drugs to combat mitochondrial dysfunction. The goal of my research program is to produce a more complete understanding of mitochondrial biology by systematically establishing the functions of orphan mitochondrial proteins and their roles within disease‐related processes. We begin by devising multi‐dimensional analyses designed to make new connections between these proteins and established pathways and processes. We then employ mechanistic and structural approaches to define the functions of select proteins at biochemical depth. This ‘systems biochemistry’ strategy is helping us address outstanding biological questions, including: Which orphan mitochondrial proteins fulfill missing steps in classic mitochondrial processes, such as the biosynthesis of coenzyme Q and the assembly of complex I, and how do they operate mechanistically? And, which resident signaling proteins direct the post‐translational regulation of mitochondrial activities? In answering these and other questions, we aim to help transform the mitochondrial proteome from a component list into a metabolic circuitry of connected functions, and to establish the biochemical underpinnings of mitochondrial dysfunction in human disease.Support or Funding InformationNIH R01DK098672 NIH R35GM131795 NIH R01GM115591 NIH P41GM108538
Death by Lipids: role of noncoding RNAs in metabolic stress
Authors: Schaffer Jean E 
Published: April 2020

Nutrient excess is linked to the pathogenesis of complications of obesity and type 2 diabetes. Fatty acid accumulation in non‐adipose tissues is initially well tolerated and leverages the nearly universal ability of cells to store fatty acids as triglycerides in lipid droplets. However, lipid overload eventually precipitates cell dysfunction through generation of toxic metabolites, activation of signaling cascades, endoplasmic reticulum stress, and oxidative stress. These pathways contribute to cell death, organ dysfunction and disease pathogenesis. Mechanisms that regulate the transition from adaptive lipid storage to initiation of lipotoxicity are not well understood. New insights have come from genetic screens in model cell culture systems. By leveraging the ability of retroviral promotor trap mutagenesis to disrupt genetic elements regardless of coding potential and using selection for resistance to palmitate‐induced cell death, our group discovered that disruption of specific small nucleolar RNAs (snoRNAs) protects against lipotoxic and oxidative stress in cultured cells. By extending our findings to in vivo models, we have demonstrated that knockdown or knockout of these snoRNAs rewires metabolism in ways that increase lipid metabolism, enhance oxidative stress tolerance, and confer resistance to lipotoxicity. Our studies highlight novel roles for snoRNAs as metabolic modulators and suggest that expression and function of this class of intronic non‐coding RNAs may have relevance to human health and disease.Support or Funding InformationNIH R01 DK064989NIH R01 DK108357
Toolbox to evaluate biological function of histone deacetylases
Authors: Fierke Carol A. 
Published: April 2020

Lysine acetylation is a post‐translational modification that is regulated by the opposing activities of histone acetyltransferases that catalyze protein lysine‐N‐acetylation, and histone deacetylases (HDACs) that catalyze removal of the ɛ‐N‐acetyl‐lysine modification. Acetylation regulates a plethora of cellular processes, and misregulation has been implicated in the development of myriad pathological states such as cancer, diabetes, aging, neurological disorders and genetic diseases. Inhibitors of metal‐dependent HDACs have been approved by the FDA for the treatment of T‐cell lymphoma and multiple myeloma. The full complement of in vivo substrates for the 11 metal‐dependent HDACs is unknown, and the functional basis for substrate selectivity is poorly defined. We are investigating the substrate sets for HDAC1, HDAC6 and HDAC8 using a number of methods, including: 1) identification of transient interactors using active‐site directed photocrosslinking covalent capture followed by mass spectrometric analysis, 2) measurement of HDAC activity with libraries of peptides and proteins coupled with the development of computational selectivity models, and 3) analysis of the role of phosphorylation and protein binding partners on substrate selectivity. These data are providing insight into the biological function of these important enzymes.Support or Funding InformationNational Institutes of Health R01‐GM‐040602
Structural Basis of Lipid Scrambling and Ion Conduction by TMEM16 Scramblases
Authors: Accardi Alessio , Falzone Maria , Lee Byoung-Cheol , Cheng Xiaolu , Khelashvili George , Weinstein Harel 
Published: April 2020

The plasma membrane of eukaryotic cells is asymmetric, with polar and charged lipids sequestered to the inner leaflet when the cell is at rest. Activation of phospholipid scramblases rapidly collapses this asymmetry and externalizes negatively charged phosphatidylserine lipids, activating extracellular signaling networks that control processes such as apoptosis, blood coagulation, membrane fusion and repair. The TMEM16 family is comprised of Ca2+‐dependent Cl− channels and phospholipid scramblases with non‐selective ion channel activity. Structural and functional analyses of TMEM16 scramblases identified a membrane‐exposed hydrophilic groove that serves as a dual translocation pathway for ions and lipids. The mechanisms underlying Ca2+‐dependent gating of TMEM16 scramblases/non‐selective channels, lipid permeation and the emergence of their ion conduction pore remain poorly understood. We used cryo‐electron microscopy to determine the structures of a fungal scramblase/non‐selective channel from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs in the presence and absence of Ca2+. These structures reveal that Ca2+ binding induces a global rearrangement of the transmembrane and cytosolic regions, resulting in opening of the lipid permeation pathway by way of rearrangements of TM4 and TM6. The structures of the scramblase/nanodisc complex in the presence and absence of Ca2+ show that the scramblases induce a pronounced distortion of the surrounding membrane: the outer leaflet is bent along the dimer axis of the protein and the membrane is thinned near the opened lipid translocation pathway thereby facilitating transbilayer lipid movement.Molecular dynamics simulations of a second closely related fungal TMEM16 scramblase, nhTMEM16, revealed that a hydrophobic lock between TM3 and TM4 is essential to maintain the open, lipid‐conductive conformation of the scramblase. MD simulations suggest that disruption of this lock favors the positioning of TM4 giving rise to a Ca2+‐bound conformation that is closed to the membrane and thus non‐conductive for lipids. Indeed, mutations at the TM3/TM4 lock severely impair lipid scrambling. Notably, these mutants retain ion channel activity, indicating that the dual activity scramblase/channel was converted into a channel only protein. The cryoEM structure of a channel‐only mutant reveal a continuous, protein‐delimited pore of sufficient size to allow ion permeation. Our results show that TM6 and TM4 are the gating elements of TMEM16 scramblases where TM6 movement is directly controlled by Ca2+‐binding and TM4 rearrangements are controlled by a hydrophobic lock between TM3 and TM4.Support or Funding InformationNIGMS 5R01 GM106717
Genome wide analysis of heparan sulfate assembly
Authors: Esko Jeffrey D. , Weiss Ryan J. , Spahn Philipp , Toledo Alejandro Gomez , Chiang Austin W.T. , Kellman Benjamin P. , Li Jing , Benner Christopher , Glass Christoper K. , Gordts Philip L.S.M. , Lewis Nathan E. 
Published: April 2020

It is well known that the arrangement and orientation of the sulfated sugar residues in heparan sulfate specify the location of distinct binding sites for protein ligands, and that binding regulates important biological processes including cell proliferation and development. Most of the enzymes involved in glycosaminoglycan biosynthesis have been cloned, purified and studied biochemically, but there is much less information regarding the regulatory mechanisms that control the spatial and temporal expression of heparan sulfate in cells and tissues. We have taken a two‐prong approach towards uncovering these control systems. In one approach, we examined the promoter regions of all genes involved in heparin/heparan sulfate assembly and uncovered a transcription factor binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR‐mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to a dramatic increase in expression of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin. Enhanced 3‐O‐sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition. Analysis of transcriptomics data showed an inverse correlation of expression of ZNF263 and HS3ST1 in mast cells and basophils compared to other (non‐heparin producing) immune cells, consistent with its role in regulation of anticoagulant heparin. In a second approach we developed a genome‐wide CRISPR/Cas9‐mediated screen. A lentiviral single guide RNA (sgRNA) library was utilized to knock down gene expression across the entire genome in a human melanoma cell line. High‐throughput screening assays were adapted to identify lentiviral‐encoded sgRNAs that induce resistance to a cytotoxin whose action depends on HSPGs and that reduce binding of HS‐dependent ligands. Top hits from the screens were characterized and categorized based on their predicted gene functions and are currently being individually validated and examined for their involvement in the regulation of HS composition.Support or Funding InformationWe thank generous support from the National Institutes of Health (R01GM33063 to J.D.E.; R21CA199292 to J.D.E. and N.E.L., R35GM119850 to N.E.L, R21 HD089067 to P.L.S.M.G.), the Fondation Leducq (to C.K.G and P.L.S.M.G.), a fellowship award to R.J.W. (K12HL141956), and generous support from the Novo Nordisk Foundation Center for Biosustainability at the Technical University of Denmark (to N.E.L.).
Leveraging Microfluidics for High‐Throughput Studies of Transcription Factor/DNA Binding
Authors: Fordyce Polly , Aditham Arjun , Horton Connor , DelRosso Nicole , Mokhtari Daniel , Markin Craig 
Published: April 2020

Gene expression is regulated primarily by transcription factors (TFs) that bind specific regulatory DNA sequences within the genome, thereby recruiting or repressing the transcriptional machinery to activate or repress transcription, respectively. However, despite decades of research, we remain largely unable to predict TF binding and/or downstream gene expression from regulatory sequence alone. Here, we leverage a microfluidic platform to yield quantitative, high‐throughput measurements of TF‐DNA binding affinities and dissociation rates for hundreds of basic helix‐loop‐helix TF variants interacting with varying DNA sequences. These data establish that sequences outside of known consensus sites can have surprisingly large effects on equilibrium binding and quantitatively map interactions across the TF‐DNA interface. We anticipate that these results will prove broadly useful for computational efforts to predict functional effects of rare mutations in precision medicine and promoter design in synthetic biology.Support or Funding InformationP.M.F. is a Chan Zuckerberg Investigator and also acknowledges a Sloan Research Fellowship for supporting this work.
Role of ECM in the Brain‐Gut Connection
Authors: Gomez-Pinilla Fernando , Yang Xia 
Published: April 2020

We sought to determine the role of extracellular matrix (ECM) proteoglycans on regulating brain function and cell metabolism. In the first series of studies, we used dietary fructose to perturbate metabolism based on evidence that fructose is a major contributor to the epidemic of diabetes and obesity. Our transcriptomic studies (Meng et al, EBiomed, 157(7), 2016) showed that the gene for the proteoglycan biglycan (Bgn) occupies a central position in a network of genes receptive to the action of fructose in the hypothalamus (main brain center of metabolic control). We pursued studies further and exposed male biglycan knockout mice (Bgn0/−) to fructose for 7 weeks, and results showed that the Bgn0/− gene perturbation compensated for a decrement in learning and memory performance in mice exposed to fructose (Ying et al., BBADIS, 1864(12) 2018). Learning and memory performance was consistent with an attenuation of the action of fructose on hippocampal plasticity markers CREB and BDNF. Bgn siRNA treatment abolished the effects of fructose on CREB and BDNF levels, in conjunction with reducing a fructose‐related increase in Bgn protein. In addition, fructose consumption perturbed the systemic metabolism of glucose and lipids, that were also altered in the Bgn0/ mice. Transcriptomic profiling of hypothalamus, hippocampus, and liver supported the regulatory action of Bgn on key molecular pathways involved in metabolism, immune response, and neuronal plasticity. Overall results underscore the tissue‐specific role of the extracellular matrix in the regulation of metabolism and brain function, and support Bgn as a key modulator of the impact of fructose across body and brain. In separate studies, we found that the gene for the proteoglycan fibromodulin (Fmod) controls a network of genes responsive to the action of brain trauma (EBiomed, 184(16) 2017). The action of Fmod (42–80 KDa) on the brain is poorly understood and rather known for affecting fibrillogenesis, cell adhesion, cytokine activity in the periphery. We found that deletion of Fmod gene mitigated the TBI‐related increase in the pro‐inflammatory marker toll like receptor 4 (TLR4), and attenuated a reduction in cell metabolic markers PGC1alpha and cytochrome oxidase II (COII), in the mouse hypothalamus after TBI. The latter suggests that Fmod deletion can protect against the cell metabolic crisis observed after TBI. In addition, given the regulatory role of the hypothalamus on peripheral metabolism, we sought to determine the effects of Fmod genetic deletion on the homeostasis of lipids in the liver on mice exposed to TBI. Results showed that the deletion of the Fmod gene protected against the action of brain trauma on key lipids and the levels of fatty acid synthase (FAS), a major lipogenic enzyme commonly involved in fatty acid processing. These results suggest a potential effect of Fmod on brain trauma outcome in brain and periphery.Support or Funding InformationR01 NS50465, R01 DK104363
Mechanisms of Wnt5a‐Ror Signaling in Development and Disease
Authors: Ho Henry , Konopelski Sara , Susman Michael , Kunz Ryan , Tan Jia , Cohen Michael , Okada Kyoko , Lamb Helen , Choi Shannon , Karuna Edith , Scales Michael , Gygi Steven , Greenberg Michael 
Published: April 2020

Wnt5a‐Ror signaling is a conserved developmental pathway that regulates morphogenetic processes during vertebral embryonic development, and dysfunction of the pathway causes a number of human disorders, including Robinow syndrome. The mechanisms of Wnt5a‐Ror signaling remain poorly understood. Using a large‐scale proteomic screen, we identified the E3 ubiquitin ligase Pdzrn3 as a new regulatory target that is degraded upon pathway activation in a beta‐catenin independent, ubiquitin‐proteasome system dependent manner. Using this discovery, we developed a flow cytometry‐based reporter to monitor Pdzrn3 abundance and delineated a signaling cascade involving Frizzled, Dishevelled, Casein kinase 1, and Glycogen synthase kinase 3 that regulates Pdzrn3 degradation. Genetic epistasis analysis suggests that Pdzrn3 resides downstream of Dishevelled and independently of Kif26b, a previously identified Wnt5a‐Ror regulatory target. Further, we discovered that Pdzrn3 degradation requires Wnt5a‐dependent phosphorylation of its C‐terminal LNX3H domain, which is conserved in several Pdzrn3 homologs and likely functions as a novel Wnt5a‐responsive domain. Collectively, this work establishes a new Wnt5a‐Ror signaling cascade involving Pdzrn3 phosphorylation and degradation.Support or Funding InformationNIH Grant 1R35GM119574