bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural and functional characterization of threonylcarbamoyl adenosine biosynthesis in bacteria
Authors: Paranagama Naduni , Luthra Amit , Bayooz Susan , Jeon Youngmin , Lyumkis Dmitry , Swairjo Manal 
Published: April 2020

tRNA, the central molecule of translation, is heavily modified post transcriptionally with noncanonical nucleosides that constitute ~83% of all RNA modifications. Modifications of the anticodon stem‐loop (ASL) of tRNA are required for translational fidelity and codon diversification. The ASL modification N6‐threonylcarbamoyl adenosine (t6A) at position 37 in ANN‐decoding tRNAs (N is any nucleobase) is universally conserved and its deficiency in humans causes renal‐neurological disease. Due to its essentiality in bacteria, the t6A pathway has emerged as a potential antibacterial target. Bacterial t6A biosynthesis begins with synthesis of the intermediate threonylcarbamoyl adenylate (TC‐AMP) from threonine, ATP and bicarbonate by the TsaC enzyme, followed by transfer of the threonylcarbamoyl moiety from TC‐AMP to A37 of substrate tRNA by the theonylcarbamoyl transfer (TCT) complex, comprised of the three proteins TsaB, TsaD and TsaE. We recently showed in T. maritima that while the TsaBD complex is the substrate binding platform responsible for TC transfer, the complex becomes inactivated after each t6A synthesis cycle, and TsaE‐dependent ATP hydrolysis “resets” the platform for multi‐turnover catalysis. However, the nature of the inactivation and reset steps remain unclear. Recent reports of a kinase activity for TsaE prompted us to investigate the role of this activity in the t6A cycle. Using a radiochemical kinase assay and mutagenesis experiments we show that T. maritima TsaE switches between a highly stable multimeric form possessing a tyrosine autokinase activity and a monomeric form lacking kinase activity and functioning as an ATPase in t6A synthesis. Further, to elucidate the structural basis of the specificity of t6A biosynthesis to ANN‐decoding tRNA substrates, we initiated cryo‐EM studies of the tRNA‐bound TCT complex. The preliminary maps reveal tRNA bound on the back surface of the TsaD subunit with its anticodon lodged in the TC‐transfer site and acceptor stem stabilized by the C‐terminal tail of TsaB, consistent with previous biochemical data. Collectively, the results provide insight into the mechanism of TC transfer, and suggest a regulatory role of TsaE that links t6A synthesis to other cellular processes.Support or Funding InformationFunding: NIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).
Simple and Affordable Kinetic Assay of Nucleic Acids by Gel Staining
Authors: Guillen Danielle , Schievelbein Mika , Patel Kushkumar , Jose Davis , Ouellet Jonathan 
Published: April 2020

In an effort to circumvent the pitfalls of using radioactive labeling to measure kinetics in undergraduate labs, a gel staining kinetics assay was developed. The IR‐3 enzyme and substrate, a single stranded DNA that cleaves another single stranded DNA only in the presence of Zn++, was used as a simple and inexpensive DNA model. The rate of the IR‐3, or D‐Zyme, was successfully determined by staining PAGE‐Urea gels with SYBR Gold and then viewing them at 302 nm with epi‐illumination. The open source software ImageJ (version 1.52K) was utilized to quantify the relative band intensities, the ratio of cleaved product to the addition of product and substrate, gave percent cleavage. When percent cleavage was plotted against time in Minitab (version 18.1, Minitab, Inc.), the rate of the reaction could then be determined by nonlinear regression curve‐fitting to the single exponential. To certify this technique, it was also tested on two other models, one being a modified version of the D‐Zyme, while the other was a known Hammer Head Ribozyme, trans RzB. It was demonstrated that the use of SYBR Gold post‐migration staining can be used to quantify RNA and DNA bands for cleavage kinetic analysis, without substrate labeling. Although this method is comparatively less sensitive than radioactive and fluorescence labeling, it has several advantages such as: i) ease of use; ii) cost effective; iii) no half‐life decay; iv) absence of substrate labeling; v) precision enabling curve‐fitting; vi) versatility with non‐structured and structured RNAs or DNAs.Support or Funding InformationMonmouth University Summer Research Program
Exploring Structural Determinants and Functional Aspects of Micron‐scale Protein Assemblies in Living Cells
Authors: Levin Tal , Seisdedos Hector Garcia , Empereur-Mot Charly , Levy Emmanuel 
Published: April 2020

Proteins are increasingly often seen as forming micron‐scale assemblies in their folded state. Such assemblies include, for example, CTP synthase, Glutamine synthase or Acetyl CoA carboxylase, all of which can form filaments under particular environmental or cellular conditions. Despite the rising prevalence of these micron‐scale structures, their structural basis and functional roles remain poorly understood at a cellular level. In particular, are there general structural principles underlying the formation of these assemblies? Is their accidental formation deleterious to cells? Do they naturally co‐localize with the quality‐control machinery of the cell? Does altering their formation impact cell fitness? To address these questions, we endogenously mutate several yeast proteins to either promote or inhibit their micron‐scale self‐assembly. We will present the results of these experiments, which provide answers to the questions posed above.Support or Funding InformationEuropean Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 819318)
Kinetic and Computational Investigations into the Origin of Metal‐Ion Specificity in Matrix Metalloproteinase‐1
Authors: Grove Laurie E. , Palladini Jacob 
Published: April 2020

Matrix Metalloproteinases (MMPs) are a family of Zn‐dependent proteases responsible for cleaving peptide bonds. MMPs have essential roles in cell proliferation, differentiation, immune responses, among many others. Human MMPs have been implicated in a number of diseases including cancer and arthritis and as such have been the focus of studies related to their roles. We are interested in exploring the high specificity for the Zn2+ ion by MMP‐1 using kinetic assays and computational modeling. Human MMP‐1 contains a classical HEXXHXXGXXH Zn‐binding domain. Both the Zn2+ and second sphere Glu219 residue are involved in the first step of catalysis – deprotonating a Zn‐coordinated water. Interestingly, the Volvox species has a proteinase (VMP3) with a modified QEXXHXXGXXH metal‐binding domain that displays enhanced activity with Cu2+ and is much less active with Zn2+. In our study, a variety of metal‐substituted samples of MMP‐1 were prepared using dialysis to remove the Zn2+ ion. Kinetic assays using a FRET substrate demonstrate less than 5% activity in all cases when Cu2+ is substituted for Zn2+. To further explore these results, we developed a series of computational models of the MMP‐1 active site substituting either Zn2+ or Cu2+ into the active site. Density Functional Theory (DFT) as implemented in the ORCA program was used to develop these models, simulating the protein environment using either solvation models or by directly including second sphere residues as part of the quantum mechanics (QM) calculation. Models were validated by comparison to experimental results, including the kinetic data and spectroscopy data in the case of the Cu2+ models. Our work shows two effects at play. First, the first sphere geometry in MMP‐1 is primed to support a tetrahedral coordination environment, which is preferred by Zn2+ whereas Cu2+ prefers a square planar coordination environment. Second, the positioning of the Zn2+‐OH2 unit in the active site positions the water for deprotonation by Glu219. In the Cu2+ models, the distortion of the Cu2+‐OH2 unit due to the less stable coordination species increases the energy associated with proton transfer. This work provides a clear example of how first and second sphere active site residues are finely tuned to maximize enzyme activity.Support or Funding InformationWentworth Institute of Technology
Cross Regulation of Phospholipid and Unsaturated Fatty Acid Biosynthesis
Authors: Dible Sarah , McDonough Virginia 
Published: April 2020

As most cellular phospholipids require unsaturated fatty acids (UFAs), we investigated the potential cross regulation of phospholipid and UFA biosynthesis. The Δ‐9 desaturase, encoded by OLE1, is the sole source of endogenous UFAs in Saccharomyces cerevisiae. Inspection of the OLE1 promoter revealed several sequences that are close matches to the regulatory sequence UASINO found in many phospholipid biosynthetic genes, which is the target of regulation by Ino2p, Ino4p and Opi1p. We examined if these proteins also play a role in regulation of OLE1 expression. Reporter gene analysis showed that when uracil prototrophy was placed under control of the OLE1 promoter, poor expression was observed in the opi1 and ino4 mutants. When lacZ was placed under control of the OLE1 promoter, reporter assays confirmed that expression was poor in the tested mutants. qPCR analysis demonstrates that the down regulation of OLE1 occurs normally in opi1, ino2 and ino4 mutants, however, its expression under growth conditions without exogenous UFAs is poor compared to wild type cells. Fatty acid analysis revealed that normal regulation of OLE1 occurred in response to unsaturated fatty acid (UFA) supplement in the opi1 mutant, indicating that post‐transcriptional regulation is still functional. Finally, the 2‐hybrid assay was used to examine potential interactions between Ole1p and Opi1p, Ino2p, Ino4p and known OLE1 regulators Mga2p and Spt23p, however no unexpected interactions were detected. This preliminary work indicates that phospholipid regulators Opi1p, Ino2p, Ino4p play a role in OLE1 expression, but not through regulation in response to UFA supplements or through Mga2p and Spt23p.Support or Funding InformationResearch reported was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM132853
Contacts and Confirmations that Regulate Hexameric Helicases at the Replication Fork
Authors: Trakselis Michael 
Published: April 2020

Duplication of the genome requires efficient, rapid, and coordinated activities of many enzymes that interact within a larger replisome complex. The hexameric helicase is at the leading front of the replisome and is a nexus of control for both the initiation and elongation phases of DNA replication. These proteins are comprised of a N‐terminal DNA binding domain and a C‐terminal motor domain that assemble into a toroidal hexameric structure. Two main superfamilies of hexameric helicases exist: the RecA family, which contain helicases from phage and bacteria, and the AAA+ family, which contain helicases from archaea and eukaryotes. Interestingly, the measured translocation polarity of 5′‐3′ or 3′‐5′, respectively, dictates the orientation of these helicases at the replication fork.Using site specific DNA cleavage experiments and presteady‐state FRET, we have unequivocally shown the then N‐terminal domain of the archaeal MCM helicase leads the way for unwinding (N‐first). The MCM hexamer itself can bind model DNA substrates in multiple conformations; however, the correct ‘N‐first’ conformation is dictated by 1) the presence of an excluded strand, 2) the ssDNA polarity (3′‐5′), and 3) the C‐terminal winged‐helix (WH). This orientation is opposite to that of the bacterial DnaB helicase which proceeds C‐first in the 5′‐3′ direction.To determine the cellular impact of the excluded strand interaction in the steric exclusion and unwinding model (SEW), we created and tested several CRISPR/Cas9 genetic mutations of E. coli dnaB that correlate with mutations on the exterior surface. These DnaB mutations alter the conformation of the hexamer creating a spectrum of constricted states that all result in faster DNA unwinding. Genetic dnaB mutations lead to decoupling within the replisome, buildup of ssDNA, activation of the SOS response, and loss of genomic integrity. Therefore, the SEW interaction and the central channel diameter is adaptive and dynamic to properly maintain the DNA unwinding within the confines of the replisome to regulate speed of DNA replication and prevent chromosomal instability.Support or Funding InformationNational Science Foundation Division of Molecular and Cellular Biosciences (NSF1613534)
Activity‐Dependent and Compartment‐Specific Neuronal Redox Dynamics
Authors: Radhakrishnan Saranya , Tantama Mathew 
Published: April 2020

Reactive oxygen species (ROS) are integral components of redox signaling in cells and mediate physiologically essential signaling across compartments. Under pathological conditions, excessive presence of ROS can lead to oxidative stress that damages cellular components and leads to cellular dysfunction and death. To understand dynamic redox changes and identify the mechanisms that disrupt redox homeostasis, we need high‐precision tools that can be used to define redox processes in live cells under physiological and pathophysiological conditions.Genetically encoded fluorescent sensors that measure cellular redox status can be targeted to specific subcellular compartments to study the redox changes in intracellular compartments during live‐cell imaging. However, it has remained technically challenging to quantitatively correlate spatially distinct redox dynamics with subcellular resolution. To address this challenge, we developed red fluorescent redox sensors that can be multiplexed with reduction‐oxidation‐sensitive green fluorescent proteins (roGFP) to image dual compartments within the same cell. Our approach utilized Förster‐type resonance energy transfer (FRET) in a spectral relay strategy that extends the fluorescence emission of roGFP into red emission wavelengths. The construct was then characterized both in solution and through dual compartment imaging in cell lines.ROS increases are ancillary to pathological processes like glutamate excitotoxicity and mitochondrial dysfunction that can propagate neuronal damage. Mitochondria help regulate the metabolic burden through the detoxification of neurotransmitters and the utilization of metabolic substrates, thereby regulating extracellular levels of glutamate. Following preliminary characterization of the sensor in cell lines, we targeted the sensors to mitochondria and cytosol in primary hippocampal neurons and monitored sensor response to oxidizing and reducing agents as well as to neuronal stimulation. Glutamate stimulation of neurons results in an increase in mitochondrial oxidation accompanied by a cytosolic reduction. Pharmacology indicates that the compartment‐specific redox responses are coupled and involve mitochondrial electron transport. Thus, using our sensors we have observed novel redox phenomena in primary neurons.
An autoinhibitory mechanism in regulating m6A‐methylation of 28S rRNA
Authors: Song Jikui , Ren Wendan , Lu Jiuwei 
Published: April 2020

N6‐methyladenosine (m6A) modification represents an evolutionarily conserved epitranscriptomic mechanism that critically regulates RNA metabolism and function. m6A is widely present in mRNA and long noncoding RNA, as well as in ribosomal RNA (rRNA) and small nuclear RNA, mediated by an array of distinct writer enzymes. However, how m6A writers attain substrate specificities remains unclear. CCHC zinc finger‐containing protein ZCCHC4 is a newly identified RNA methyltransferase that specifically mediates the m6A modification on the site 4220 of 28S rRNA, which in turns impacts translation, cell proliferation and tumor growth. To provide mechanistic insights into the ZCCHC4‐mediated m6A RNA methylation, we performed structural characterization of the enzyme‐cofactor complex of human ZCCHC4. Our work reveals that the regulatory domains and the methyltransferase (MTase) domain pack into an integrated fold, thereby creating a multi‐domain RNA‐binding platform. Importantly, the MTase domain adopts an autoinhibitory conformation, in which the active site is occluded from RNA binding by an inter‐domain loop segment. Mutational and enzymatic analyses further indicate that this loop segment directly engages substrate recognition, providing an autoinhihitory mechanism by which the activity of ZCCHC4 is coupled with specific substrate binding. In addition, analysis of the substrate sequence and structure suggests a role of the stem‐loop structure within substrate in governing the substrate specificity. Together, our study uncovers an autoinhibitory mechanism that ensures specific interplay between ZCCHC4 and the RNA substrate, reminiscent of what was seen with the DNA methylation writers.Support or Funding InformationNIH 1R35GM119721
ApoE genetic variation is associated with differential susceptibility to dyslipidaemia and type 2 diabetes in black Africans
Authors: Tanyanyiwa Donald Moshen , Buthelezi Philani Ernest , Dandara Collet , Bhana Sandeep Amrat 
Published: April 2020

BackgroundDyslipidaemia in type 2 diabetes mellitus (T2DM) is characterized by a biochemical lipid triad of low HDL‐cholesterol, raised triglycerides, and small, dense low‐density lipoprotein (LDL) particles. Studies on ApoE genetic variation influence in the development of dyslipidaemia in African diabetic patients are few.Aims and ObjectivesThis study aimed to evaluate the role of genetic variation in ApoE coding gene on dyslipidaemia in South African diabetic patients.MethodsTwo hundred and forty‐four (n=244) participants were retrospectively recruited from the Baragwanath diabetic clinic. These comprised of two groups: (i) dyslipidaemic, and (ii) non‐dyslipidaemic (controls). The dyslipidaemic group was further divided into three groups according to; i) high cholesterol only; ii) high triglycerides only; and, iii) both high cholesterol and high triglycerides (referred to as the mixed group). Ethical clearance was obtained from the University of Cape Town and Baragwanath/Wits University. Genetic characterisation of ApoE was accomplished using polymerase chain reaction/restriction fragment length polymorphism (RFLP), confirmed by Sanger sequencing.ResultsOf the 244 participants, 165 were dyslipidaemic while 79 were not. A significant (p <0.0001) finding in this study is that ApoE2/3 dyslipidaemic participants had a distribution of only 5.3%, compared to 20.6% among the non‐dyslipidaemic participants. There was a low frequency distribution, 1.8% of among ApoE2/4 among the diabetic participants. There a significant association between HbA1c and age (p=<0.008). TC (p=0.00092), LDL (p=0.0184) and triglycerides (p=0.0175) were strongly associated with poor glycaemic control. Both LDL (p=0.0174 and HDL (p=0.0072) were associated with age.ConclusionsThe study showed the effects of ApoE genetic variation on the dyslipidaemia seen in black South African diabetic participants. The study showed that diabetic dyslipidaemia is genetically influenced by Apo E. ApoE2/3 DM patients had a reduced risk of developing dyslipidaemia while ApoE2/4 was associated with reduced risk for T2DM. Therefore, exploration of possible underlying genetic predisposition in dyslipidaemic T2DM patients who respond poorly to standard therapy is recommended.Support or Funding InformationDepartmental support
Fluorescence Localization of the Conjugation Machinery of Bacillus subtilis
Authors: Chen Sirui , Dame Haley B. , Berkmen Melanie B. 
Published: April 2020

Conjugation, or mating, is the transfer of DNA from a donor to a recipient cell through conjugation machinery. Conjugation contributes to bacterial genome innovation and the spread of virulence and antibiotic resistance genes between bacteria. DNA is transferred during conjugation through a multi‐protein DNA translocation channel, classified as a Type IV secretion system (T4SS), embedded in the membrane. Our work focuses on characterizing the conjugation machinery of the integrative and conjugative element ICEBs1 of Bacillus subtilis. Previously, we identified several protein components of the ICEBs1 conjugation machinery, including the bitopic membrane protein ConB and the peripheral membrane ATPase ConE. We have used fluorescence microscopy to observe the locations of ConB and ConE inside cells. A fusion of ConE to green fluorescent protein (GFP) localizes to the membrane, predominantly at the cell poles. Here, we show that a fusion of ConB to the red fluorescent protein mCHERRY localizes similarly to ConE‐GFP. Consistent with ConE lacking predicted transmembrane segments, ConE‐GFP’s localization to the membrane requires ConB. In contrast, localization of ConB‐mCHERRY to the membrane does not depend on other conjugation proteins. Current studies are focused on which domains of ConB are required for its own localization as well as the localization of ConE.Support or Funding InformationThis research was funded by Suffolk University and an NSF‐RUI grant to M. Berkmen.
Molecular signatures of neuroinflammation induced by α‐synuclein aggregates in microglial cells
Authors: Sarkar Souvarish , Dammer Eric , Malovic Emir , Seyfried Nicholas , Tansey Malu , Kanthasamy Anumantha , Rangaraju Srikant 
Published: April 2020

Alpha‐synuclein aggregates (α‐synAgg) are pathological hallmarks of Parkinson’s disease (PD) and other synucleinopathies that induce microglial activation and immune‐mediated neurotoxicity, but the molecular mechanisms of α‐synAgg‐induced immune activation are poorly defined. We performed quantitative proteomics by mass spectrometry coupled with PCR, immunohistochemical and functional validations studies to define the molecular characteristics of alpha synuclein mediated microglial activation. In mouse microglia, α‐synAgg induced robust pro‐inflammatory activation (increased expression of 864 genes including Irg1, Ifit1 and Pyhin) and increased nuclear proteins involved in RNA synthesis, splicing and anti‐viral defense mechanisms. Conversely, α‐synAgg decreased expression several proteins (including Cdc123, Sod1 and Grn), which were predominantly cytosolic and involved in metabolic, proteosomal and lysosomal mechanisms. Pathway analyses and confirmatory in vitro studies suggested that α‐synAgg partly mediates its effects via Stat3 activation. As predicted by our proteomic findings, we verified that α‐synAgg induces mitochondrial dysfunction in microglia. 26 proteins differentially expressed by α‐synAgg were also identified as PD risk genes in genome‐wide association studies (upregulated: Brd2, Clk1, Siglec1; down‐regulated: Memo1, Arhgap18, Fyn and Pgrn/Grn). We then validated progranulin (PGRN) as a lysosomal PD‐associated protein that, relative to the vast upregulation of activated microglia, is decreased in nigral microglia in post‐mortem PD brain compared to non‐disease controls, congruent with our in vitro findings.ConclusionTogether, proteomics approach both reveals novel molecular insights into α‐syn‐mediated neuroinflammation in PD and other synucleinopathies.Support or Funding InformationWork supported by Emory Alzheimer’s Disease Research Center Grant P50 AG025688, Alzheimer’s Association (SR #37102), NINDS (K08‐NS099474‐1) and Emory Neuroscience NINDS Core facilities (P30 NS055077) to S.R.; NINDS (NS100090) and NIEHS (ES026892) to A.G.K