bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural characterization of BEX3 (brain expressed X‐linked) (755.1)
Authors: Almeida Marcius , Raymundo Diana , Cabral Katia Maria 
Published: June 2021

The aim of this work is to characterize BEX3 at molecular level. BEX3 is a p75NTR‐associated protein that mediates apoptosis in response to Nerve Growth Factor, by interacting with the intracellular death domain of p75‐NTR. Besides a small number of studies validating the interaction of BEX3 and p75‐NTR in vivo, very little is known about the interaction of these proteins. For instance, the 3D structure of BEX3 is still uncharacterized. These proteins have been expressed heterologously at high level in the soluble fraction of Escherichia coli, and them purified by nickel affinity and gel filtration chromatography. The protein was characterized by solution nuclear magnetic resonance, circular dichroism, fluorescence spectroscopy, atomic force microscopy, small‐angle X‐ray scattering, partial proteolysis and partial cross‐linking. Surface plasmon resonance binding experiments using Biacore X system reveled binding and dissociation of BEX3/p75‐NTR with Kd within nanomolar range, which fits to an one‐site binding curve (R2=0.9999). The 3D structure of BEX3 has high content of flexibly disordered regions in addition to residual alpha‐helix structure and folded hydrophobic core at its C‐terminus. We discuss the capacity of this protein to form high‐order oligomers to avoid aggregation.Grant Funding Source: Supported by FAPEJ and CNPq.
A conserved ER‐membrane complex facilitates phospholipid exchange between the ER and mitochondria (758.5)
Authors: Lahiri Sujoy , Chao Jesse , Tavassoli Shabnam , Wong Andrew , Young Barry , Loewen Christopher , Prinz William 
Published: June 2021

The objective of this study is to find the mechanism of lipid exchange between the endoplasmic reticulum (ER) and mitochondria. Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the ER to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER‐membrane proteins complex (EMC) has decreased phosphatidylserine (PS) transport from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER‐mitochondria tethering complex called ERMES (the ER‐mitochondria encounter structure) are inviable, and PS transfer to mitochondria is dramatically reduced, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER‐mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER associated with mitochondria, even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that the EMC interacts with the mitochondrial TOM complex protein, Tom5, and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.Grant Funding Source: NIDDK intramural research program
The mitochondrial Ca2+‐independent phospholipase A2 becomes active in parallel to OPA1 cleavage (760.1)
Authors: Rauckhorst Adam , Pfeiffer Douglas 
Published: June 2021

Accumulation of depolarizing amounts of Ca2+ or Sr2+, as well as chemical uncoupling have been shown to activate a mitochondrial Ca2+‐independent phospholipase A2 (iPLA2). Data generated in isolated mitochondria utilizing various electron transport chain inhibitors indicate that neither redox status of particular electron transport chain complexes nor ROS production are primary regulators of the iPLA2; however, loss of membrane potential (∆Ψ) is shared by all of the treatments that produce activation. Therefore, we hypothesized that the iPLA2 is primarily regulated by ∆Ψ. Here we report that the dynamin‐like GTPase OPA1 is cleaved from a long form to a short form in parallel to iPLA2 activation. OPA1 cleavage is known to inhibit mitochondrial fusion generating a pool of fragmented mitochondria, which are thereafter targeted for degradation by agents such as PINK1 and Parkin, and for eventual removal by autophagocytosis. Thus, both iPLA2 activation and OPA1 cleavage may contribute to initiating the turnover of poorly functioning mitochondria: a process that has been previously proposed to involve the mitochondrial permeability transition.Grant Funding Source: Supported by the Ellie Kovalcik Foundation
Activation of survival signals in melanoma cells in response to glucose deprivation (766.9)
Authors: Cammarata Garrett , Calianese David , Best Charlie , Wan Yinsheng 
Published: June 2021

Cancer’s use of alternate cellular pathways is necessary in order to proliferate and expand so aggressively but may make it possible to target using certain cancer therapies. We studied the effects that lowered glucose concentrations have on important cancer characteristics in WM266‐4 Melanoma cells. Changes to the cells included observable effects like morphology alteration and inhibition of growth as well as modification to internal signaling pathways. The cytoskeleton, being important for its interaction with insulin containing vesicles, altering of GLUT transporter translocation and glucose metabolism related enzymes, was disrupted after 48 hours which can be seen by the cells decreasing dendricity. Inhibition of growth was found to be correlated with decreasing glucose concentrations in medium, as was depletion of nutrients and changes in pH of the extracellular environment. Reduction in pH could be a mechanism of the cancer cells to proliferate and survive, the result of heavy production of lactic acid caused by increased rate of glycolysis, or both. Levels of p‐S6, p‐AMPK, p‐ERK and p‐MEK were shown to be increased by glucose deprivation at 6 hours showing that the cancer cells are fighting back in order to survive. S6 ribosomal protein as well p‐ACC decreased with glucose deprivation at 48 hours while p‐AMPK increased showing high levels of stress and eventually cellular shutdown. Finally, GLUT1 transporter expression as well as survivin, an anti‐apoptotic molecule, was shown to be increased at 24 hours under glucose deprivation. Collectively, our data suggest that the effects of glucose deprivation on melanoma are important in understanding the way these cells uptake their nutrients and may provide for therapeutic treatments in the future.Grant Funding Source: Supported by INBRE research grant
Elucidating biosynthesis of the outer membrane lipid phthiocerol dimycocerosate by Mycobacterium tuberculosis PapA5 (768.13)
Authors: Himmler Megan , Bommineni Gopal , Metz Thomas , Tonge Peter , Seeliger Jessica 
Published: June 2021

Mycobacterium tuberculosis (Mtb) has a complex, asymmetric outer membrane consisting of mycolic acids and other unique lipids. Phthiocerol dimycocerosate (PDIM) is a wax ester lipid found in the outer membrane, at the interface between the bacterium and host, where it has been implicated in Mtb virulence. My work focuses on gaining a better understanding of how PDIM biosynthesis is regulated in Mtb by studying PapA5, an acyltransferase responsible for acylating phthiocerol with mycocerosic acid. We show using a series of synthetic substrates that PapA5 has a preference for longer alkyl chains. We demonstrate under novel biphasic reaction conditions that PapA5 is capable of catalyzing two successive acylation reactions, consistent with its proposed role in modifying both hydroxyls on the phthiocerol moiety. The introduction of bulky amino acid side chains in the putative substrate‐binding pocket of PapA5 yielded partial or complete inhibition of product formation. These data help confirm the location of the substrate‐binding channels. Furthermore, the introduction of a salt bridge or a disulfide bond to constrain a hypothesized flexible helix led to partial inhibition of product. These data support a role for conformational flexibility in ligand binding and/or product release. Finally, in vitro phosphorylation of PapA5 altered product yield, demonstrating a possible mechanism of regulation for PDIM biosynthesis.Grant Funding Source: Supported by a Stony Wold‐Herbert Fund award to JCS
Novel aspects of iron sulfur cluster biosynthesis in sulfate reducing bacteria (768.17)
Authors: Butland Gareth , Saini Avneesh , Trotter Valentine , Price Morgan , He Jennifer , Kuehl Jennifer , Wetmore Kelly , Liu Nancy , Zane Grant , Fels Samuel , Juba Thomas , Shatsky Maxim , Arkin Adam , Chandonia John‐Marc , Wall Judy , Deutschbauer Adam 
Published: June 2021

Iron sulfur (FeS) cluster containing proteins are involved in processes targeted by environmentally relevant stressors. These stress conditions, for sulfate reducing bacteria (SRBs) such as Desulfovibro vulgaris Hildenborough (DvH) and Desulfovibrio alaskensis G20, would include exposure to oxygen (and oxygen radicals), the presence of N‐oxyanions such as nitrate and nitrite, and exposure to potentially toxic metals such as selenium, cobalt and chromium. Significantly, FeS cluster damage in key enzymes such as pyruvate‐ferredoxin oxidoreductase (PFOR) has been proposed as the source of inviability of SRBs and other anaerobes when encountering oxygen. Some FeS cluster biosynthesis key factors have been identified in these SRBs by sequence homology. We are characterizing the effect of these mutations on key FeS dependent marker enzymes, including PFOR, formate dehydrogenase and dissimilatory sulfite reductase, in order to determine relative contributions of these factors to FeS cluster biosynthesis. Concurrently, we have also performed fitness profiling of a comprehensive G20 mutant pool under stress conditions known to damage FeS enzymes. We have observed mild fitness defects for the G20 Dde_1444/1445 (sufBC) mutant strains, consistent with reduced marker FeS enzyme activities of the DvH sufB mutant. Interestingly, fitness data has indicated that the sole cysteine desulfurase mutant in G20 (nifS; Dde_3078) displays pleotropic defects, but remains viable, contradicting current dogma regarding FeS cluster biosynthesis.Grant Funding Source: Supported by the U. S. Department of Energy under Contract No. DE‐AC02‐05CH11231
Reprogramming the chemodiversity of terpenoid cyclization by remolding the active site contour of epi‐isozizaene synthase (769.5)
Authors: Harris Golda , Li Ruiqiong , Chou Wayne , Aaron Julie , Litwin Kevin , Cane David , Christianson David 
Published: June 2021

The class I terpenoid cyclase epi‐isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi‐isozizaene as the predominant sesquiterpene cyclization product, along with at least 5 minor sesquiterpene products. The active site contour of EIZS serves as a template that enforces a particular substrate conformation and chaperones carbocation intermediates through a well‐defined mechanistic sequence. We have engineered the active site of EIZS by remolding its hydrophobic contour through site‐directed mutagenesis in order to decipher the code that relates the three‐dimensional contour of the active site with the products generated by the enzyme. Significantly, the remolded cyclization template in EIZS mutants reprograms the reaction cascade not only to adjust the proportions of products generated by the wild‐type enzyme, but also to generate completely new products of diverse structure. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100‐fold losses of catalytic activity, suggesting that product diversity, and not necessarily catalytic efficiency, is a critical driver in the evolution of terpenoid biosynthesis.Grant Funding Source: Supported by GM56838
Role of active site residues Ser237 and Arg276 in CTX‐M‐14 β‐lactamase substrate binding and catalysis (774.6)
Authors: Adamski Carolyn , Cardenas Ana Maria , Brown Nicholas , Horton Lori , Palzkill Timothy 
Published: June 2021

Extended spectrum β‐lactamases (ESBLs) pose a significant threat to public health because of their ability to confer resistance to third generation cephalosporins such as cefotaxime. CTX‐M‐14 β‐lactamase is one of the most widespread ESBL enzymes among antibiotic resistant bacteria and was used in this study to identify residues that facilitate resistance to cephalosporins. We investigated the roles of Ser237 and Arg276 in CTX‐M‐14 β‐lactamase using site‐directed mutagenesis, kinetic analysis and X‐ray crystallography. We show that, together, Ser237 and Arg276 residues are key determinants of cefotaxime hydrolysis by the CTX‐M‐14 β‐lactamase. X‐ray structures of S70G:S237A:R276A and S70G:S237A variants of CTX‐M‐14 alone and in complex with cefotaxime revealed changes in the orientation of the aminothiazole ring on the cefotaxime side chain suggesting that these residues form a cooperative network which allows cefotaxime to assume a more buried position in the active site thereby facilitating catalysis. Finally, simultaneous substitution of CTX‐M‐14 positions 237 and 276 was sufficient to transform the substrate profile of CTX‐M‐14 to that of a non‐ESBL class A β‐lactamase indicating these residues make critical contributions to the cefotaximase activity of CTX‐M family of enzymes.Grant Funding Source: Supported by NIGMS T32 GM008280 and NIH R01 AI32956
A bioinformatic analysis of the vertebrate Twist protein family (778.5)
Authors: Rodríguez Yacidzohara , Gonzalez‐Mendez Ricardo , Cadilla Carmen 
Published: June 2021

Twist protein family members are expressed in different tissues during early stages of embryogenesis and their presence is essential for proper development and survival. Though the highly conserved basic helix‐loop‐helix (bHLH) and carboxy‐terminal domains of Twist proteins have received a lot of attention, the amino‐terminal region and the evolution of such proteins merit further study, particularly among mammals. We analyzed the conservation of the amino‐terminal region of different vertebrate Twist proteins by sequence comparisons. We identified two putative de novo motifs (SSSPVSP and SEEE), specifically in mammals. In addition, a number of amino acid substitutions present in the nuclear localization signals (NLSs) of a few species demonstrate that there could be other possible residues influencing the nuclear localization of such proteins, with the G residue of the second NLS being a potential target. By phylogenetic analysis, we investigated the origin and evolution of the two paralog twist proteins. First, a gene duplication event occurred to produce the two twist paralogs. Second, among the two twist paralog branches, twist1 is the most distant in vertebrates, suggesting it evolved first due an abrupt increase of substitutions in a short period of time due to functional divergence. Also, in mammals, twist1 seems to have a higher rate of evolution than twist2 since only twist1 sequences contain two glycine‐rich motifs not present in twist2. Our findings shed light on the relationship of twist1 and twist2 paralogs among mammals in vertebrates.Grant Funding Source: Supported by NIH grants No. T36GM008789 and P41 RR06009
Mass spectrometry as a tool for structural biology: crosslinking‐MS and native‐MS methods to study the RNA Pol II ‐ Mediator holoenzyme (778.6)
Authors: Trnka Michael , Robinson Philip , Kornberg Roger , Burlingame A 
Published: June 2021

The Mediator complex plays a central role in transcriptional regulation by relaying gene specific regulatory signals to the general transcriptional machinery. The yeast holoenzyme between mediator and RNA pol II consists of 33 subunits totaling over 1.5 MDa in size. The large size precludes atomic resolution crystallography of the entire assembly. Hence hybrid methods of structure determination are necessary, integrating various techniques including: crystallography of stable subassemblies, cryoEM of the intact complex, and mass spectrometry (MS). Mass spectrometry based structural techniques including chemical crosslinking and native‐MS provide spatial restraints that guide and constrain the modeling process. Crosslinking measurements map interacting protein‐protein surfaces through the identication of amino acid residues that have been covalently modified by heterobifunctional reagents. Native MS permits the determination of stably associated assemblies of proteins in the gas phase. We focus on the development of these new MS based technologies including the design of robust bioinformatics strategies, enrichment methods, and reagents for optimizing mass spectrometric analysis of crosslinked peptides, as well as deconvolution algorithms and sample preparation for native MS analysis. We discuss the application of these methods to determining the structure of the Mediator holoenzyme.Grant Funding Source: Supported by: NIH P41GM103481
Relative quantification of the effect of blood meal iron on protein expression in the ovaries of Aedes aegypti (778.8)
Authors: Geiser Dawn , Li Wenzhou , Breci Linda , Winzerling Joy 
Published: June 2021

Diseases transmitted by mosquitoes are estimated to kill more than 2 million people annually. The vector for dengue, Aedes aegypti (yellow fever mosquito), is found in the desert Southwest of the continental United States. One approach to limiting the spread of disease is to reduce the numbers of mosquito vectors. Our area of research is iron metabolism. Our long term goal is to use molecular techniques to alter the expression of iron related proteins in mosquitoes to reduce mosquito fecundity and survival. Our hypothesis is that female mosquitoes require iron in the blood meal as an essential nutrient to aid in egg development. Although the iron is necessary for fecundity it is also highly oxidative and toxic in biological systems unless the mineral is strictly regulated to mitigate its oxidative potential. We are currently analyzing tissue‐specific, iron regulated proteins of the mosquito ovary 24 h post‐blood meal fed on artificial blood meal diets supplemented with no iron or low iron, or a porcine blood meal (high iron). Measurement of the blood meal iron effect on ovarian protein expression was carried out using Tandem Mass Tags (TMT) which permits simultaneous identification and relative quantification of proteins during LTQ Velos Orbitrap LC‐MS/MS analysis. This research will contribute to our understanding of mosquito development in general, as well as ovarian iron metabolism.Grant Funding Source: Supported by funds from the Arizona Ag Experiment Station and CALS
Congenital disorder of glycosylation caused by a mutation in SSR4, the signal sequence receptor 4 protein of the TRAP complex (789.3)
Authors: Losfeld Marie‐Estelle , Ng Bobby , Kircher Martin , Buckingham Kati , Turner Emily , Eroshkin Alexey , Smith Joshua , Shendure Jay , Nickerson Deborah , Bamshad Michael , Freeze Hudson 
Published: June 2021

Congenital Disorders of Glycosylation (CDG) are a family of genetic disorders affecting the glycosylation pathway or the trafficking of glycoproteins. Nearly 50 genes causing CDG have been described, but many patients biochemically diagnosed with CDG do not have mutations in known genes. Among our CDG patients, we identified by exome saequencing, in three patients, new variants of the X‐linked SSR4 gene which encodes a protein of the heterotetrameric translocon‐associated protein (TRAP) complex. Two of these variants results in the absence of SSR4 expression. In these cases, we observed that expression of other TRAP complex proteins was also reduced. The glycosylation marker Glyc‐ER‐GFP was used to confirm the underglycosylation in fibroblasts from patients. Over‐expression of the wild type SSR4 allele partially restores glycosylation of the marker and expression of the other members of the TRAP complex. This is the first evidence that the TRAP complex, which binds to the oligosaccharyltransferase complex, is directly involved in N‐glycosylation.Grant Funding Source: Supported by the National Institutes of Health grant [R01DK55615] and “The Rocket Fund”.
Genetics and virulence role of the classical group A Streptococcus Lancefield antigen (790.2)
Authors: Cole Jason , van Sorge Nina , Kuipers Kirsten , Henningham Anna , Aziz Ramy , Kasirer‐Friede Ana , Lin Leo , Berends Evelien , Davies Mark , Dougan Gordan , Zhang Fan , Dahesh Samira , Shaw Laura , Gin Jennifer , Cunningham Madeleine , Merriman Joseph , Rooijakkers Suzan , Malley Richard , Walker Mark , Shattil Sanford , Schlievert Patrick , Choudhury Biswa , Nizet Victor 
Published: June 2021

All strains of group A Streptococcus (GAS), a leading cause of infection‐related mortality worldwide, express group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N‐acetylglucosamine (GlcNAc) side chain. The immunodominant GlcNAc epitope of GAC is the basis of all rapid diagnostic testing for GAS infection, but a function for this eponymous antigen in disease pathogenesis has never been proposed. Here, we identify the genetic basis of the GAC GlcNAc side chain and generate an isogenic mutant for analysis of its role in virulence and immunogenicity. Loss of the GlcNAc epitope on GAC attenuated GAS virulence in two animal infection models, in association with increased sensitivity to killing by whole blood, neutrophils, and platelet‐derived antimicrobials. Antibodies generated against the modified GAC structure promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge in mice after passive immunization. Our results demonstrate how functional insight into the classical Lancefield antigen has implications for GAS disease pathogenesis and vaccine development.Grant Funding Source: National Institutes of Health; National Health and Medical Research Council of Australia
Disruption of the nuclear membrane by the antimicrobial peptide LL‐37 leads to the formation of neutrophil extracellular traps (793.1)
Authors: Neumann Ariane , Völlger Lena , Nerlich Andreas , Meerloo Timo , Gallo Richard , Nizet Victor , Naim Hassan , von Köckritz‐Blickwede Maren 
Published: June 2021

Neutrophil extracellular traps (NETs) have been described as a fundamental innate immune defense mechanism. During formation of NETs the nuclear membrane of mammalian cells is disrupted by a yet unknown mechanism. The cathelicidin LL‐37 has been shown to bind and damage membranes deficient in cholesterol and sphingomyelin such as bacterial membranes. Consequently, we hypothesized that LL‐37 could potentially damage the nuclear membrane, which contains reduced amounts of cholesterol, and thereby facilitates the formation of NETs by activated neutrophils.Different concentrations of LL‐37 were used to stimulate primary blood‐derived neutrophils. The percentage of NET‐releasing cells was calculated using immunofluorescence microscopy and revealed that LL‐37 is able to facilitate NET‐formation. Cells treated with LL‐37 showed a clear disruption of the nuclear membrane by electron microscopy and confocal immunofluorescence microscopy. Accordingly, when blocking the cleavage and subsequent activation of endogenous LL‐37, NET formation was significantly diminished. Biochemical assays utilizing a random LL‐37‐fragment library indicated that the NET‐induction is mediated by the hydrophobic character of the peptide.In conclusion, this study demonstrates a novel role of the antimicrobial peptide LL‐37 in host immune defence: the disruption of the nuclear membrane and subsequent formation of NETs.Grant Funding Source: German Research Council (DFG)