bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pre‐Clinical Evaluation of Drug Induced Proteolysis of MLL‐Fusions in Acute Leukemia
Authors: Looi-Somoye Remi , Cantilena Sandra , de Boer Jasper 
Published: April 2020

The mixed‐lineage leukemia (MLL) gene is often susceptible to chromosomal rearrangements in pediatric leukemias. The resulting chimeric proteins are a product of reciprocal translocations between the gene and various fusion partners. In MLL‐rearranged leukemia, these fusion proteins drive leukemogenesis by interfering with transcriptional elongation to deregulate target gene expression. MLL fusion proteins, indicative of a more aggressive disease, are responsible for a large proportion of acute infant leukaemias. Therapeutic methods to target oncogenic fusion proteins, such as PML‐RARA, have proven pharmacologically achievable and highly successful. For this purpose, our lab has established a drug screening program using in cellulo bio‐luminescence with the objective of repositioning drugs that cause MLL‐fusion protein degradation. Compounds that produced a 30% reduction in MLL fusion protein expression via two different screening methods, luminescence and western blotting, were considered positive hits. In this present study I validate and characterize one of these positive hits. Upon treatment of MLL‐rearranged cell lines, the compound was capable of ablating MLL fusion protein expression resulting in the down regulation of target gene HOXA10. Furthermore, treatment caused loss of leukaemic stem cell activity via induction of cell death and inhibition of self‐renewal capacity. To further analyze the mechanism of this compound, RNA‐sequencing and Gene Set Enrichment Analysis will be performed to identify gene sets associated with the disease treated phenotype. Overall this study identifies a potential repurposed therapeutic for the treatment of MLL‐rearranged leukaemias.Support or Funding InformationThis work was supported by the UCL Child Health Research Studentship.
Neurotoxic Effect of Paracetamol on Female Rats: Role of Antioxidant Treatment and Prevention
Authors: Al-Qirim Tareq , Jasim Suhair , Shawaqfeh Bara’a 
Published: April 2020

Paracetamol (Acetaminophen) is the most common nonprescription analgesic and antipyretic drug used. It can be found in different pharmaceutical dosage forms such as syrup, capsule, suppositories and intravenous (I.V) infusion solution. The intake frequency and the dose of paracetamol usually varied during pregnancy with a tendency toward higher doses in the last trimester. The effect of Paracetamol overdose was extensively studied on hepatocyte and nephrocyte of laboratory animals. The aim of this study is to focus on the effect of paracetamol overdose on Neurotoxicity (within Hippocampus, Cerebellum, and Olfactory Bulbs) of Paracetamol in the last trimester of pregnant rats. And this will be done through studying oxidative stress markers, the biochemical and antioxidant tests that indicate the presence of Hepatotoxicity and Nephrotoxicity, along with the protective effect of Vitamin E as antioxidant in pre‐ and post‐single toxic dose administration. Twenty female pregnant Wistar rats (average weight 200 gm ± 10) at gestational day 18 (GD18) were used. The pregnant rats were divided into four groups; the first (control) group received a 0.5 ml p.o of corn oil. The second group (paracetamol) group received a 3000 mg/kg p.o paracetamol dissolved in corn oil. The third (E + paracetamol) group received a 3000 mg/kg paracetamol one hour after 300 mg/kg p.o vitamin E. The fourth (paracetamol + E) group received a 3000 mg/kg paracetamol one hour before 300 mg/kg p.o vitamin E dissolved in corn oil. Twenty‐four hours After Paracetamol administration the rats were anesthetized and the brain, the liver, the kidney, and the blood were collected. Various biochemical tests were performed to show the effect of paracetamol overdose on liver including Alanine aminotransferase (ALT), Aspartate aminotransferase (AST). Additionally, the effect of paracetamol on Blood urea nitrogen (BUN) and Creatinine levels were determined to detect nephrotoxicity, and the results showed significant elevation within the paracetamol treated group and levels were restored to the normal levels with vitamin E treated groups. Uric acid (UA) and superoxide dismutase (SOD) were used to detect the oxidative stress within the Liver, the Kidney, the Hippocampus, the Cerebellum, and the Olfactory Bulbs.Support or Funding InformationThis research is supported form Al‐Zaytoonah University of Jordan
A Genetic Approach to Display and Dissect the Cancer‐Associated O‐Glycoepitome
Authors: Clausen Henrik , Nason Rebecca , Mandel Ulla , Narimatsu Yoshiki 
Published: April 2020

A hallmark of cancer is aberrant protein O‐glycosylation with expression of truncated immature O‐glycans including the classical Tn, STn, and T pancarcinoma antigens. The truncation of O‐glycans not only present epitopes recognized by the natural low affinity antibodies to the TTn glycan haptens, which are the basis for the polyagglutination phenomena, but also expose composite aberrant O‐glycopeptide epitopes that are not normally expressed and can elicit high affinity IgG antibodies. Several examples of monoclonal antibodies with high selectivity for truncated O‐glycopeptide epitopes, which in effect selectively target the cancer form of O‐glycoproteins, have been produced in the past. Studies demonstrate that such epitopes serve as highly cancer‐specific targets potentially amenable for potent T‐cell engaging strategies, however, the current insight into the abundance and distribution of aberrant O‐glycopeptide epitopes and the optimal glycoforms for these remain limited. To systematically explore antibody epitopes available in the cancer‐associated O‐glycoproteome, we have developed a cell‐based platform for display and production of human O‐glycoproteins with homogeneous truncated glycoforms. This platform includes CHO and HEK293 cells stably engineered with homogeneous glycosylation capacities for the Tn, STn, and T glycoforms, and a panel of reporter expression constructs containing segments of human mucins and mucin‐like domains from O‐glycoproteins for display of these with different O‐glycans. An overview of these efforts will be presented.Support or Funding InformationDanish National Research Foundation (DNRF107) and the Lundbeck Foundation
Role of Ascorbic Acid in Skeletal Muscle Regeneration
Authors: Yatsu Tomofumi , Takino Yuka , Ishigami Akihito 
Published: April 2020

Total amount of L‐ascorbic acid (AsA) in human skeletal muscle is approximately 40%. As satellite cells, muscle specific stem cells, are responsible for providing new myoblast cells during the muscle regeneration process. Myoblast cells construct myofibers in the regenerative process including proliferation and differentiation. AsA is known to prevent scurvy helping dioxygenases for the prolines hydroxylation in collagen. It has been reported that AsA accelerated Ten‐Eleven Translocation (Tet) methylcytosine dioxygenase‐mediated generation of 5‐hydroxymethylcytosine (5hmc) from 5‐methylcytosine. We have previously reported that AsA deficiency causes skeletal muscle atrophy and deterioration in physical performance in senescence marker protein 30 knockout (SMP30‐KO) mice. In this study, we showed that AsA deficiency causes a delay of skeletal muscle regeneration in SMP30‐KO mouse which is unable to biosynthesis of AsA. Moreover, AsA administration after skeletal muscle injury recovered it regeneration. In vitro experiment, AsA treated C2C12 mouse myoblast cells showed higher cell proliferation rate, mRNA expression levels of myogenic differentiation markers such as myogenin and myomaker, and fusion index compared to untreated cells. Finally, we examine the 5hmc levels in AsA‐treated and untreated C2C12 cells. The 5hmc levels was higher in AsA‐treated cells than untreated cells. These results suggest that AsA induced skeletal muscle regeneration by enhancing the myoblast proliferation, differentiation, and DNA demethylation.Support or Funding InformationGrant‐in‐Aid for Early‐Career Scientists. Funding Agency: Japan Society for the Promotion of Science. Project number: 19K19822
Piezo1 activation gains traction
Authors: Pathak Medha 
Published: April 2020

A major unanswered question in biology is how mechanical forces are generated, detected and transduced by cells to impact biochemical and genetic programs. Our work is aimed at uncovering the mechanical principles at play in cells and tissues using novel molecular, imaging and bioengineering tools. Here we present insights gleaned from non‐invasive approaches to measure and manipulate mechanical signals in native cellular conditions. We find that the mechanically‐activated ion channel Piezo1 transduces cell‐generated traction forces to regulate lineage choice of neural stem cells. We show that actomyosin‐based cellular traction forces generate spatially‐restricted Piezo1 Ca2+ flickers in the absence of externally‐applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their Ca2+ flicker activity is enriched in regions proximal to force‐producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca2+ flickers allow spatial segregation of mechanotransduction events, and that mobility allows channel molecules to efficiently respond to local mechanical stimuli.Support or Funding InformationNIH grants R01 NS109810 and DP2 AT010376
Functional Assembly of the Mitochondrial Protein Transport Machinery
Authors: Alder Nathan , Peixoto Pablo , Modak Arnab 
Published: April 2020

The vast majority of proteins that reside within mitochondria are encoded in nuclear DNA, synthesized on cytosolic ribosomes, and imported into the organelle. Within the mitochondrion reside several types of complex machineries that mediate the sorting, translocation and integration of incoming polypeptides based on their targeting information. Among these translocases, the TIM23 Complex mediates the import and biogenesis of most resident proteins, which are synthesized with cleavable amino‐terminal targeting sequences. The TIM23 Complex is a dynamic multi‐subunit assembly that serves versatile functions in mediating the transport of soluble proteins across the mitochondrial inner membrane as well as the lateral integration of membrane proteins. The multifunctional nature of this complex may arise from different modular states that it can assume based on its component subunits. Using a host of biophysical approaches, we have investigated the substrate‐ and energy‐dependent structural dynamics of the central subunit, the voltage‐gated channel Tim23. We have also analyzed the lipid interactions that mediate membrane associations with the receptor subunit, Tim50, as well as channel‐receptor interactions. To investigate the structure and function of the membrane‐sorting modular form of TIM23, we have utilized a reductionist approach in which five component subunits are purified from a heterologous expression system and reconstituted into a biomimetic model membrane. These subunits include the polytopic channel proteins Tim23 and Tim17, the Tim50 receptor, and additional membrane proteins Tim21 and Mgr2. The innovation of this system derives from the fact that multiple independently purified membrane proteins can form a functional complex spontaneously when reconstituted with the proper topology in a bilayer of synthetic lipids. This reconstituted complex provides a minimal system for the direct analysis of its channel features, its energetic requirements for mediating membrane protein integration, and the factors that mediate assembly, including lipid requirements. It also provides a highly purified system for structural analysis.Support or Funding InformationThis work was supported by NIH Grant 1R01GM113092 (to N.N.A.) and PSC‐CUNY Grant PSC‐CUNY 626860050 (to P.P.)
Constraining evolution → avoiding drug resistance: lessons from viruses
Authors: Schiffer Celia 
Published: April 2020

Drug resistance negatively impacts the lives of millions of patients and costs our society billions of dollars by limiting the longevity of many of our most potent drugs. Drug resistance occurs when heterogeneity exists within a genome and selection weakens inhibitor binding but maintains the biological function of the target. Genome diversity occurs both from transcriptional infidelity and the specific activity of human APOBEC3’s.To avoid drug resistance, the structure at atomic resolution and evolutionarily constraints on its variation is required. This rationale was derived from our lab’s experience with substrate recognition both of human APOBEC3s and of antiviral drug targets. In particular we have acquired a rich and versatile experimental dataset of viral proteases altered by the selective pressures of inhibitors. With this data we are integrating alterations in both the protein sequence and the inhibitor with changes in potency and we correlate this data to our co‐crystal structures and our strategy of parallel molecular dynamics (pMD) to elucidate specificity and the molecular mechanisms of drug resistance.pMD is a strategy we have developed to collectively analyze a series of MD simulations of similar yet distinct molecular complexes to decipher conformational and dynamic differences responsible for changes in molecular recognition. We perform pMD simulations on complexes with varying protein sequence and ligand identity to unravel structural and dynamic properties that underlie coupled changes in molecular recognition and resistance. We have applied this pMD strategy to protein‐ligand complexes for series of natural substrates, inhibitors, and protease mutations. Combined with experimental potency data, we determine which physical properties or molecular interactions are key to molecular recognition for a given system.These changes in structure and dynamics dictate the interdependency of molecular mechanism of resistance and areprincipals that are generally applicable to other quickly evolving diseases where drug resistance is quickly evolving.Support or Funding InformationNIH P01 GM109767, R01 AI085051, R01AI150478
Structural Insights into TRPV Channel Gating
Authors: Moiseenkova-Bell Vera 
Published: April 2020

The transient receptor potential vanilloid 5 (TRPV5) channel is a highly calcium selective ion channel that regulates systemic calcium homeostasis by acting as a critical gate for calcium reabsorption in the kidney. Human polymorphisms of this channel have exemplified the importance of TRPV5 in disorders of calcium homeostasis, but atomic level information regarding this channel had remained unknown. To address this gap, we utilized cryo‐electron microscopy (cryo‐EM) to understand the gating and modulation of TRPV5. Moreover, this newly obtained structural information coupled with structure‐based virtual screening allowed us to uncover novel inhibitors of TRPV5 and revealed unique mechanisms of TRPV5 inhibition. These studies have thus expanded the base of understanding of TRPV5 at the atomic level and laid the foundation for future rational drug design.Support or Funding InformationNIGMS R01GM103899; NIGMS R01GM129357
Cardiolipin‐dependent Carriers
Authors: Claypool Steven , Senoo Nanami , Kandasamy Selvaraju , Ogunbona Oluwaseun , Baile Matthew , Lu YaWen 
Published: April 2020

The phospholipid, cardiolipin, has pleiotropic structural and functional roles that are collectively essential for mitochondrial biology. Yet, the molecular details of how this lipid supports the structure and function of proteins and protein complexes is poorly understood. To address this property of cardiolipin, we use the mitochondrial ATP/ATP carrier as a model. Here, we have determined that cardiolipin is critical for both the tertiary and quaternary assembly of the major yeast Aac isoform, Aac2, as well as its conformation. Notably, these structural roles provided by cardiolipin are separable. Additionally, we show that multiple copies of Aac2 engage in shared complexes that are largely dependent on the presence of assembled respiratory complexes III, IV, or respiratory supercomplexes. Intriguingly, the assembly state of Aac2 is sensitive to its transport‐related conformation. Collectively, these results significantly expand our understanding of the numerous structural roles provided by cardiolipin for mitochondrial membrane proteins.Support or Funding InformationThis work was supported by grants from the National Institutes of Health (R01HL108882 to S.M.C and Biochemistry, Cellular, and Molecular Biology Program Training Grant T32GM007445 to M.G.B. and Y.L.), pre‐doctoral fellowships from the American Heart Association (15PRE24480066 to O.B.O., 10PRE3280013 to M.G.B. and 12PRE11910004 to Y.L.), and a post‐doctoral fellowship from the Uehara Memorial Foundation (to N.S.).
Getting Hedgehogs Where They Need to Go: Cleavage Activates Dispatched for Sonic Hedgehog Release
Authors: Ogden Stacey K. 
Published: April 2020

Hedgehog ligands activate an evolutionarily conserved signaling pathway that provides instructional cues during tissue morphogenesis, and when corrupted, contributes to developmental disorders and cancer. The transmembrane protein Dispatched is an essential component of the machinery that deploys Hedgehog family ligands from producing cells, and is required for Hedgehog morphogen gradient formation and signaling to long‐range targets. Despite this crucial role, regulatory mechanisms controlling Dispatched activity remain largely undefined. We reveal that Dispatched is activated by Furin‐mediated cleavage at a conserved processing site in its first extracellular loop. CRISPR/Cas9 gene editing was used to generate transgenic mice harboring cleavage site mutation in the endogenous Dispatched locus. Homozygous animals show neuronal patterning phenotypes and decreased viability, confirming a requirement for Dispatched processing in vivo. As such, Furin serves as a crucial post‐translational regulator of Dispatched activity in Hedgehog‐producing cells during tissue morphogenesis.Support or Funding InformationThis research was supported by funding from the National Institute of General Medical Sciences grants R01GM114049, R35GM122546.
Roles of Notch Glycoslation in Signaling
Authors: Stanley Pamela 
Published: April 2020

Notch receptors are single pass transmembrane glycoproteins with an extracellular domain (ECD) comprised of many epidermal growth factor‐like (EGF) repeats. Specific Ser and Thr residues in numerous EGF repeats have an attached O‐glycan initiated by fucose, glucose, or GlcNAc, and elongated with 1–3 more sugars. Notch receptors signal in response to the binding of Notch ligands to Notch ECD. Notch signaling regulates cell fate decisions in many organs during development and differentiation, and its dysregulation leads to developmental disorders and cancer. A point mutation that precludes the addition of a single O‐fucose glycan to EGF12 in the NOTCH1 ligand binding domain alters Notch signaling differently depending on genetic background. In a mixed genetic background a mild effect on T and B cell development is observed, whereas in the C57BL/6J background, lack of the EGF12 O‐fucose glycan is embryonic lethal. Lunatic (LFNG), Manic (MFNG) and Radical (RFNG) Fringe are GlcNAc transferases that modify Notch EGF repeats which already carry a fucose. We are investigating roles for each Fringe in T and B cell development using mice lacking two or three Fng genes. We are also identifying individual roles for Fng genes using a co‐culture assay containing OP9 stromal cells expressing one of the four Notch ligands, DLL1, DLL4, JAG1 or JAG2 and bone marrow hemopoietic cells from mice expressing all Fng genes, no Fng genes (tKO) or a single Fng gene. The combined results show that the O‐fucose glycans on NOTCH1 are key regulators of the strength of NOTCH1 signaling induced by canonical Notch ligands.Support or Funding InformationNational Institutes of Health grant RO1 GM106417National Institutes of Health grant PO1 CA13330
Watching a Fine‐tuned Molecular Machine at Work: Structural and Functional Studies of the 26S Proteasome
Authors: Martin Andreas , Jonsson Erik , Greene Eric , Lopez-Alfonzo Erika , Htet Zaw , Gates Stephanie 
Published: April 2020

The degradation of ubiquitin‐tagged proteins by the 26S proteasome is a highly complex process with several well‐coordinated sub‐steps, including ubiquitin recognition, deubiquitination, substrate engagement by the AAA+ motor, mechanical unfolding, translocation, and proteolytic cleavage. Our recently developed FRET‐ and fluorescence‐based assays, relying on the labeling of proteasome‐incorporated unnatural amino acids, allow us to dissect the kinetics and coordination of individual processing steps, and study the coupled conformational changes of the proteasome. Single‐molecule measurements provide exciting details about the conformational dynamics of the proteasomal regulatory particle and how ubiquitin binding, substrate engagement by the AAA+ motor, or the presence of certain receptor subunits affect the switching between substrate‐free and substrate‐processing states, which are furthermore controlled through specific interactions between the ATPase hexamer and the proteasomal lid subcomplex.FRET‐based assays reveal how substrates are propelled through the central channel of the proteasome and progress through the various stages of degradation, including a translocation stall during deubiquitination and the backtracking of substrate upon failure to remove ubiquitin chains prior to substrate entry into the AAA+ motor. Our mutational studies thereby provide important new insight into the mechanisms of substrate engagement and the contributions of individual ATPase subunits depending on their position in the vertical spiral‐staircase arrangement of the AAA+ hexamer. By using substrates with various architectures, we also gained a better understanding of how a substrate’s geometry and ubiquitin modifications affect the degradation rate, and how the proteasome may prioritize its substrates in the cell.Support or Funding InformationHoward Hughes Medical Institute; NIH‐NIGMS (R01‐GM094497)
tRNA Quality Control: Mechanisms, Evolution, and Implications for Human Disease
Authors: Phizicky Eric 
Published: April 2020

In all organisms tRNAs are subject to numerous post transcriptional modifications, imparting them with properties to accurately and efficiently execute their critical roles in translation. Modifications in and around the anticodon loop generally affect translation, whereas modifications in the tRNA body often affect tRNA folding or stability. Lack of modifications frequently results in growth and translation defects in the budding yeast Saccharomyces cerevisiae and in neurological disorders or mitochondrial diseases in humans, but the mechanisms by which these defects occur are in many cases not known. Our lab focus is to understand the exact roles of selected modifications implicated in human disease, by genetic and biochemical analysis of their roles in S. cerevisiae and in the evolutionarily distant fission yeast, Schizosaccharomyces pombe, to understand if and to what extent these roles have evolved in different eukaryotes, including humans. We will present results describing the conservation and evolution of the roles of two modifications, highlighting interactions with a widely conserved stress response pathway.Support or Funding InformationNIH NIGMS grant GM052347