bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of N‐acetyl cysteine on myofibroblast phenotype expression by immortalized keratinocytes, Ker‐CT‐Ras (731.5)
Authors: Webb Jessica , Vaughan Melville , Black Morgan , Gainer Sonnie
Published: June 2021

Recent research activity has focused on the tumor stroma. Tumor stroma are typically connective tissues containing fibroblasts and myofibroblasts. These cells are required for the wound healing processes of the body. There is evidence that myofibroblast presence in tumor stroma leads to poor prognosis. Mechanical tension, one of three key factors, enhances differentiation of myofibroblasts.

In vitro carcinomas can form through a pathway which involves the up‐regulation of the ras protein. Precancerous keratinocytes lead to two types of carcinomas. They take on properties of fibroblasts and metastasize, spreading into the dermis. Fibroblasts generate tension in the dermis during the wound healing process. Our experiment focuses on precancerous keratinocytes and their journey into the dermis, which we model using an in vitro stress‐relaxed collagen matrix.

We set up Ker‐CT‐Ras lattices void of fibroblasts and generated contraction data. With Ker‐CT‐Ras as with fibroblasts, transforming growth factor beta (TGF‐beta) increases tension generating ability and up‐regulates production of stress fibers. N‐acetyl cysteine (NAC) in the lab was shown to block tgf‐beta signaling in fibroblasts. Here we focus on the effect of NAC on Ker‐CT‐Ras. Additional data presents the comparative tension‐generating ability of fibroblast lattices (DP‐147‐H‐Tert) and co‐culture lattices comprised of the two. Data taken from a monolayer (coverslip) model visually describes the structural properties of the myofibroblast phenotype. Overall, we found that response of Ker‐CT‐Ras to these signals is similar to that of fibroblasts.

Grant Funding Source: Supported by CURE‐S‐STEM, OK‐LSAMP, and RCSA grants
De novo identification of non‐coding RNAs in aging male germline stem cells (732.8)
Authors: Luk Alfred , Ng Shuk Han , Lee Tin‐Lap
Published: June 2021

Aging is a complex time‐dependent biological process that takes place in every cell and organ, eventually leading to degenerative changes that affect normal biological functions. In the past decades, the number of older parents has increased significantly. While it is widely recognized that advanced maternal age poses higher birth defect risk due to egg aging, the effect of advanced paternal aging remains largely overlooked. Non‐coding RNAs are generally classified as non‐ protein‐coding RNAs have emerged as a novel player in many developmental processes. However its role in advanced paternal aging are unknown. Here we show a de novo approach to annotated unique lncRNA transcripts associated with male germline stem cell aging. Based on previous mice aging model consisting of 6‐day, 21‐day, 60‐day and 8‐month old animals. We identified a set of age‐dependent lncRNA transcripts that have not been annotated previously. Preliminary analysis on RNA structure and protein binding suggested the transcripts involved in a wide variety of developmental functions, including proliferation, adhesion and cell death. The findings are expected to substantially change the concepts of lncRNA function in developmental, reproductive and stem cell biology.
Encapsulation of antibiotics in poly(ethylene glycol)‐block‐poly(L‐lactide) for delivery in dentin tubules during root canal treatment (733.4)
Authors: Haseeb Ridwan , Lau Michael , Rodriguez Lucas , Montagner Franciso , Palmer Kelli , Stefan Mihaela , Rodrigues Danieli
Published: June 2021

Aim: To encapsulate an antibiotic in oral applications with PEG/PLA‐block copolymer into microparticles for infected dentin tubules. To maintain drug release for extended periods to prevent bacterial regrowth after root canal treatment.

Methodology: Drug encapsulation was carried out through an oil‐water emulsion‐solvent evaporation method. In summary, the PEG/PLA copolymer and the oral drug were dissolved in an oil phase (dichloromethane), which was combined with an aqueous (polyvinyl alcohol and de‐ionized water). The solution was stirred, centrifuged, washed, and lyophilized. Particle size was determined using digital microscopy. Antimicrobial effectiveness was assessed in vitro by placing small amounts of encapsulated particles on bacterial agar plate cultures and monitoring growth inhibition.

Results: Encapsulated particles ranged in size from 300 nm to 1.0 m. A 2 week bacterial inhibition test showed that the particles exhibited zones of inhibition between 3 mm and 5 mm.

Conclusion: The proposed method with the PEG/PLA copolymer encapsulates the oral antibiotic producing particles with size distribution that may penetrate the dentinal tubules (2.5 m in diameter). Bacterial inhibition tests showed that the particles inhibited bacterial growth after 2 weeks. Ongoing bacterial inhibition tests will determine the release profile of the microparticles.
Mechanism of mycobacterial recombination mediator proteins function in homologous recombination and strand annealing (735.12)
Authors: Korolev Sergey , Ryzhikov Mikhail , Gupta Richa , Glickman Michael
Published: June 2021

Recombination mediator proteins (RMPs) are important for genome stability in all organisms and support two reactions: the recombinase binding to single‐stranded (ss)DNA to initiate homologous recombination (HR) and strand annealing. Both reactions are inhibited by ssDNA binding proteins (e.g. gp32, SSB, RPA). RMPs promote DNA transfer from SSB to recombinase or to anneal DNA. In spite of two ubiquitous functions, RMPs lack sequence and structural homology. Previously, we demonstrated that E. coli RMPs, RecOR, do not dismiss SSB from ssDNA, but form the complex with SSB altering the conformation of bound ssDNA. RecO binds SSB and the complex promotes DNA annealing or RecA loading in the absence or presence of RecR, respectively. Surprisingly, mycobacterial RecO does not interact with SSB. Yet, both proteins form complex on the shared ssDNA substrate in both pathways.

In mycobacteria, RecFOR deletion reduces clastogens resistance to the same degree as a deletion of the major homologous recombinase RecA. Chromosomal DNA break repair assay surprisingly revealed that all three proteins are involved in both pathways: HR and annealing. Here we will describe biochemical studies of DNA annealing and strand exchange reactions supported by mycobacterial RecFOR. The selection of one or the other pathway is dictated by the availability of recombinase or complimentary strand and the stoichiometry of the DNA‐protein complexes. We believe that the intermediate complex of RMP with SSB and ssDNA represents a common critical step in reactions supported by all RMPs during HR and strand annealing DNA repair pathways in all organisms.
RNA binding protein hnRNP‐U coordinates repair of radiation‐induced damage clusters in human genome via its Ser59 phosphorylation (736.8)
Authors: Dutta Arijit , Mitra Sankar , Hegde Muralidhar
Published: June 2021

Ionizing radiation (IR), widely used in diagnostic procedures and cancer therapy, induces complex clusters of damage in the genome that include DNA double‐strand breaks (DSB) and much higher levels of closely spaced oxidized bases and single strand‐breaks (SSB). While overt DSBs are primarily repaired via non‐homologous end joining (NHEJ), oxidized bases and SSBs are repaired via base excision repair (BER)/ SSB repair pathway. However, how these multiple repair pathways are co‐ordinated at IR‐induced damage clusters is poorly understood. Here we demonstrate temporal control of NHEJ vs. BER, co‐ordinated by RNA binding protein hnRNP‐U, which was recently implicated in regulating BER for enhancing the activities of NEIL1/NEIL2 glycosylases. We found Ku, a key NHEJ protein inhibits early BER proteins (NEILs, OGG1 & APE1) at the damage clusters, preventing repair of oxidized bases at close proximity. hnRNP‐U, also recruited at the repair site cannot overcome Ku inhibition initially due to its phosphorylated state (at S59 by DNA‐PK in response to IR). Subsequently, hnRNP‐U dephosphorylated after NHEJ completion relieves Ku inhibition and facilitates BER initiation. This differential ability of unmodified vs. S59‐P hnRNP‐U is due to its phosphorylation‐dependent decrease in affinity for the DNA glycosylases. These studies provide a molecular mechanism for sequential repair of IR‐induced DSBs followed by BER of proximal oxidized bases, where hnRNP‐U acts as a ‘molecular switch’. We propose that such hierarchical repair of IR‐induced damage is important in maintaining genomic fidelity.

Grant Funding Source: Supported by USPHS grant R01 CA158910 and HMRI
The roles of differential branches of ER stress signaling in determining cell fate of autophagic hepatoma cells (737.2)
Authors: Ye Hui‐Lan , Chen Xing‐Zhou , Zhang Guo , Yin Xiu‐Xiu , Chen Li , Zhang Fa‐Can
Published: June 2021

Autophagy is a conserved lysosomal degradative pathway in eukaryotic cells and its dual effects on cell fate are controversial. The present study was designed to clarify the switching effect of IRE1 activity on ER stress‐induced autophagic cell fate and monitor the dynamic changes of autophagic processing. Firstly, by Micro Cellular Imaging and Analysis System, the fluorescent signals were dynamically recorded in HepG2 cells which were pretransfected with mRFP‐EGFP‐LC3 plasmid. With this system, cytosolic yellow puncta indicated the formation of autophagosome, while the red fluorescence puncta indicated the turnover of autolysosome in these cells. In the presence of tunicamycin (Tm) (5µg/mL) for 24h, it was found that the number of yellow puncta was markedly elevated in HepG2 cells. Interestingly, not only the intracellular yellow puncta but also red puncta were significantly increased in these cells exposured to thapsigargin (Tg) (500nM). Secondly, by western blotting, both Tm and Tg evidently induced an increment of IRE1 protein, an early attenuated branch of ER stress. However, another critical ER stress signaling molecule PERK was found to persistently increase in these cells in response to Tg, but not Tm. Thirdly, inhibiting the IRE1 activity by siRNA substantially decreased the LC3II conversion, and dramatically promoted the Tg‐induced apoptosis of HepG2 cells with about 40%. Consistently, Tg‐induced red puncta signal was apparently enhanced with IRE1 siRNA treatment. Taken together, these results suggested that IRE1 as well as PERK, the hallmarks of ER stress signaling, importantly govern the autophagic cellular outcome, which may serve as a novel potential therapeutic target for HCC.

Grant Funding Source: Supported by NSFC Grants 881172260, 30630145, 81360077 & 2010GXNSFC013020 to G.Z.
Impaired lysosome function can induce autophagy via suppression of mTOR activity (738.1)
Authors: Yang Zuolong , Guo Ne , Khambu Bilon , Zhang Hao , Liu Peiqing , Yin Xiao‐ming , Li Min
Published: June 2021

Autophagy is an evolutionarily conserved mechanism by which cytoplasmic material is degraded in the lysosomal compartment to fuel starving cells and eliminates intracellular pathogens. Autophagy can be activated via MTORC1 downregulation by amino acid deprivation and by certain chemicals. Lysosome is the degrading machine for autophagy, but has also been linked to MTORC1 activation through the Rag GTPase pathway. In this study, classical lysosome inhibitors, such as chloroquine, E64d and pepstatin A, were found to be able to inhibit mTORC1 in a Rag‐dependent manner. All these lysosome inhibitors were able to activate events associated with early autophagy biogenesis represented by ATG16L1 and ATG12 puncta formation, indicating a consequence of autophagy activation following MTORC1 suppression. Our data suggested a general link from lysosome function to the Rag‐MTORC1 signaling axis and autophagy activation. Thus the lysosome is not only required for autophagic degradation, but also affects autophagy activation. Lysosomal inhibitors can have a dual effect in suppressing autophagy degradation and in initiating autophagy.

Grant Funding Source: Supported by Guandong Provincial Natural Science Foundation, China (S2013010015876)
Effects of O‐GlcNAcylation on insulin signaling on the level of insulin receptor substrate 1 (739.13)
Authors: Hu Ping , Kim Min‐Sik , Pandey Akhilesh , Hart Gerald
Published: June 2021

β‐O‐Linked N‐acetylglucosamine(O‐GlcNAc) modification (O‐GlcNAcylation) is a dynamic posttranslational modification on Ser/Thr residues and increased O‐GlcNAc is associated with insulin resistance. Insulin receptor substrate 1(IRS1) acts as an adaptor protein transferring signals from insulin to downstream PI3K and Akt. It is O‐GlcNAcylated and highly phosphorylated. Here we mainly focused on how O‐GlcNAcylation affects insulin signaling on IRS1 level. We found that insulin signaling is regulated through pharmacologically modifying the two key enzymes’ activity: O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA). In addition, we showed phosphorylation status of IRS1 changed accordingly by vertical isoelectrical focusing and two‐dimensional gel electrophoresis. We are performing Stable Isotopic Labeling by Amino Acids in Cell Culture (SILAC) on 3T3‐L1 to detect phosphorylation changes on IRS1 as well as other signaling proteins. In addition, We mapped two O‐GlcNAc sites by CID/ETD‐MS/MS, Ser635 and Ser1001. Ser635 is a main phosphorylation site associated with type II diabetes. We found Cycling rate of O‐GlcNAc on Ser635 is fast, phosphorylation and O‐GlcNAcylation compete at Ser635 in an insulin‐responsive manner. Ongoing studies are testing how O‐GlcNAc on Ser635 affects insulin signaling in 3T3‐L1 and whether O‐GlcNAc of Ser635 affects phosphorylation level of nearby sites. These data suggests O‐GlcNAcylation on IRS1 plays an important role in regulating insulin signaling.

Grant Funding Source: NIH R01CA42486, R01DK61671; N01‐HV‐00240; P01HL107153, R24DK084949
An in vivo approach to the discovery and characterization of cellular mechanisms regulating microRNA‐mediated gene silencing (744.3)
Authors: Browning Austin , Rees Katherina , Depner Mary , Sadino Julie , Reinke Catherine
Published: June 2021

Our goal is to determine the cellular mechanisms that regulate microRNA (miRNA)‐mediated gene silencing. MiRNAs are non‐coding RNA molecules that interact with target‐gene messenger RNA transcripts via complementary base pairing, and silence target gene expression via interaction with the miRNA‐induced silencing complex (miRISC). Silencing is accomplished through translation block, mRNA degradation, or a combination of these mechanisms. A forward genetic screen was carried out to identify requirements for miRNA‐mediated silencing in Drosophila melanogaster. miRNA function was assayed using Green Fluorescent Protein (GFP)‐based reporters of gene silencing. The GFP transgenes contain miRNA binding sites, and are regulated by endogenous miRNAs. We have genetic evidence demonstrating roles for both Regena (NOT2), a subunit of the CCR4‐NOT deadenylase complex, and the protein kinase dAkt1, in miRNA‐mediated gene silencing. Further studies have implicated insulin signaling in general and specific components of the insulin‐signaling pathway in particular in the regulation of miRNA activity. We are investigating the necessity of these genes in miRNA‐mediated gene silencing at various stages of Drosophila development. Investigation of the composition, abundance, and localization of known components of the silencing machinery is revealing distinct mechanisms for the regulation of silencing.
Characterization of Celf1 function in mouse lens cell lines (746.1)
Authors: Dang Christine , Siddam Archana , Lachke Salil
Published: June 2021

Although the human genome encodes ~1500 RNA binding protein (RBP) encoding genes, mutations in only ~20 are associated with human disease. We used a novel bioinformatics approach iSyTE (integrated Systems Tool for Eye gene discovery) to identify a new RBP, Celf1 (CUGBP Elav (embryonic lethal, abnormal vision)‐Like Family member 1), in the lens that is potentially associated with cataract. Celf1 contains three conserved RNA Regulatory Motifs that function in sequence‐specific binding to target RNAs. To develop a new resource to study Celf1 function in lens cells, we generated and characterized Celf1‐knockdown (KD) mouse lens epithelial cell lines. We first selected the mouse lens epithelial cell line 21EM15 as it expresses several critical lens‐expressed genes and thus recapitulates important aspects of lens biology. We infected 21EM15 cells with lentiviral particles carrying inhibitory short hairpin (sh)RNAs against mouse Celf1 mRNA and isolated infected cells based on their acquired resistance to puromycin. Celf1‐KD was confirmed by reduced Celf1 expression in Western blotting. We are presently characterizing the effect of Celf1‐KD on distinct classes of RNA granules (e.g. Stress granules and Processing bodies) involved in post‐transcriptional regulation. In sum, we describe development of a new Celf1‐KD lens cell line as a unique resource for gaining novel functional insights of an RBP in lens biology.

Grant Funding Source: Supported by Pew Charitable Trusts
Binding of RNase R to ribosomes: comparison of non‐stop mRNA and REP sequence‐containing message (748.2)
Authors: Liang Wenxing , Deutscher Murray
Published: June 2021

RNase R, a 3' to 5' exoribonuclease able to act on most RNAs including those with extensive secondary structure, is subject to a complex regulatory mechanism. Most RNase R in exponential phase cells is sequestered on ribosomes, where it is stable. In contrast, the free form of the enzyme is turned over rapidly, presumably because it is deleterious to cells due to its promiscuous action on essential RNA molecules. We previously showed that RNase R binding to ribosomes is stimulated by non‐stop mRNA leading to its participation in trans‐translation. We show here that RNase R binding to ribosomes is also stimulated by messages containing intergenic REP sequences suggesting that degradation of these messages utilizes a similar mechanism to non‐stop mRNA decay. Interestingly, the stimulation of RNase R binding is lost when the REP sequence is moved further away from the translation termination codon and the amount of protein produced from the message is elevated. These data show that as with non‐stop mRNA, other messages which result in ribosome stalling prior to normal termination also lead to RNase R binding and likely the induction of trans‐translation.

Grant Funding Source: Supported by National Institutes of Health Grant GM16317
Characterizing the interaction between protein phosphatase 2A and the mRNA decay protein Tristetraprolin (748.4)
Authors: Wagner Allison , Nava Raul , Nickel Colin , Chase Ian , Clement Sandra
Published: June 2021

Post‐transcriptional and post‐translational processes play key roles in regulating rapid changes in gene expression by altering the activity of mRNA binding proteins on their target transcripts. The zinc‐finger mRNA binding protein TTP activates the destruction of mRNAs encoding cytokines, growth factors, and pro‐inflammatory proteins by binding to AU‐rich elements (AREs) located within the 3’ untranslated region (UTR) and recruiting mRNA decay enzymes to these targets. In response to stimuli such as a bacterial infection, the p38 MAPK activated kinase MK2 phosphorylates TTP and reduces its ability to stimulate mRNA turnover by blocking decay enzyme association. While phosphorylation of TTP has been well‐characterized, much less is known about how this process is reversed to ensure a timely but transient burst of gene expression. In order to better understand the mechanism by which TTP is dephosphorylated, we observed both the dephosphorylation status of TTP as well as PP2A heterotrimer protein levels in lipopolysaccharide (LPS) stimulated RAW 264.7 murine macrophage cells via Western blot analysis. TTP levels were highest 12 hours after LPS exposure, with dephosphorylation beginning approximately16 hours after LPS induction. Interestingly, PP2A‐C levels increased approximately five‐fold during TTP dephosphorylation. These results suggest that dephoshporylation may be regulated in part by modulation of PP2A levels. In order to better understand the mechanism by which PP2A gains access to dephosphorylate and reactivate TTP, we are currently testing the ability of TTP to associate with PP2A in both macrophages as well as in human embryonic kidney (HEK 293) cells stably transfected with wild‐type or deletion mutant forms of TTP. The results of these experiments will provide a greater understanding of the sequence of events that attenuate inflammatory responses by dephosphorylating TTP and reactivating mRNA decay.
Post‐transcriptional regulation of interleukin‐3: roles of AREs, HuR and p38 (749.5)
Authors: Hernandez Marimar
Published: June 2021

Interleukin‐3 (IL‐3) is a pro‐inflammatory cytokine that is secreted by T‐cells upon stimulation, although its mRNA is constitutively expressed, suggesting that is regulated at this level. Our group has shown that the IL‐3 3’UTR is responsible for repressing its expression, being the Adenosine/Uridine‐Rich Elements (ARE) region mainly responsible for this repression. Chimeric luciferase constructs harboring either wild‐type IL‐3 3’ UTR or different mutations on its ARE region were transfected on Jurkat cells and the reporter activity was measured upon T‐cell stimulation. We observed that when the nonamer ARE is interrupted, the 3’UTR is not able to elicit the same response as the wild type 3’UTR. We carried out EMSAs using protein extracts from Jurkat cells at 6, 12 and 24hrs after activation and identified HuR as an ARE‐Binding Protein able to bind the IL‐3 3’UTR AREs. HeLa cells were co‐transfected with plasmids over‐expressing HuR and the luciferase construct which harbors the IL‐3 3’UTR. Luciferase activity and mRNA levels decreased when HuR was over‐expressed. Since p38 has been shown to increase pro‐inflammatory cytokine secretion and is known to phosphorylate HuR, we assessed its possible role in the regulation of IL‐3. After pretreatment of Jurkat cells with the p38 inhibitor SB202190, IL‐3 mRNA levels were significantly decreased upon T‐cell stimulation suggesting that p38 is indeed involved in the post‐transcriptional regulation of IL‐3.

Grant Funding Source: Supported by U54 CA96297; NIGMS MBRS RISE R25GM061838
Cryo‐electron microscopic structure of SecA bound to the 70S ribosome (752.1)
Authors: Bhushan Shashi
Published: June 2021

SecA is an ATP‐dependent molecular motor pumping the secretory and outer membrane proteins across the cytoplasmic membrane in bacteria. SecA associates with the protein conducting channel, the hetrotrimeric SecYEG complex, in a so‐called post‐translational manner. Although, SecA can form functional dimers and high‐resolution crystal structures exist of both the monomer and dimer, the true oligomeric state of SecA remains controversial. We have determined the cryo‐electron microscopy structures of SecA bound to the ribosome. Our results show that not only a monomeric SecA binds to the ribosome but also two copies of SecA can be observed which form an elongated dimer. Results will be discussed in the context of a direct co‐translational role of SecA during protein translocation.
Core‐spanning hydrophilic residues lend stability and preserve functionality in β‐helical protein HpmA265 (752.5)
Authors: Glasgow Evan
Published: June 2021

In the present work, the bacterial β helical hemotoxin HpmA has been cloned and expressed in truncated form (HpmA265) and subject to several structural experiments. Folded (active) HpmA265 can rescue the hemolytic function of denatured, full‐length HpmA in vitro by direct interaction, and this phenomenon is quantified with a hemolytic assay. In attempt to correlate functionality with structure, circular dichroism spectroscopy has been used in conjunction with guanidine hydrochloride titrations to assess the structural/functional contributions of key hydrophilic residues in the protein’s core. Native HpmA265 was found to have a folding energy of ∆Gº = ‐50.2 ± 2.7 kJ/mol. Ala or Phe substitutions were made either at Q125 or Y134, and mutant folding energies were ‐25.0 ± 2.4 kJ/mol (Q125A), ‐48.0 ± 6.5 kJ/mol (Y134A), ‐32.4 kJ/mol (Q125F), and ‐45.4 ± 4.5 kJ/mol (Y134F). The data suggest more substantial perturbation by mutation of Q125 than of Y134. This idea complements the graphically‐predicted contribution of Q125 versus Y134, in that Q125 coordinates with an embedded water molecule in a core‐spanning hydrogen bond network and appears to be held in the proper orientation by the Y134 hydroxyl group. Additional networked residues will be assessed in this way. A greater understanding of the β helix fold may contribute to understanding of similar proteins, such as β amyloid and bacterial adhesion factors.

Grant Funding Source: Supported by NSF‐RUI 105435 (to TW)
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.
Purification of cortactin using anion exchange chromatography (755.2)
Authors: Priester Jacob , Kruchten Anne
Published: June 2021

Cortactin is an actin‐binding protein that has been shown to be involved in cellular migration and metastases in cancer. Bacterially expressed and purified cortactin protein is often used in in vitro assays to examine cortactin’s role in promoting cell migration via actin remodeling. Cortactin has a theoretical molecular weight of 60 kDa, however using SDS‐PAGE analysis the protein runs as two bands of molecular weights 80 kDa and 85 kDa suggesting that cortactin has an unusual protein folding pattern. Our current lack of understanding of cortactin structure limits our ability to determine the role of cortactin in facilitating motility phenotypes. To elucidate the forms of cortactin produced from bacterially expressed and purified cortactin protein, we used a two‐step purification system including affinity purification and anion exchange chromatography. After analysis with non‐denaturing polyacrylamide gel electrophoresis, we found cortactin protein from different anion chromatography elution fractions did not separate to similar locations on the gel across all fractions. We hypothesize that the variations in the bands are a result of different folding patterns of cortactin protein in what was once thought of as a homogenous protein pool. When testing the role of cortactin in mediating cell migration, these folding differences may have significant effects on the results of functional assays such as actin polymerization or sedimentation.
Optogenetic manipulation of cAMP in mitochondrial supcompartments (756.1)
Authors: O'Banion Colin , Lawrence David
Published: June 2021

The cyclic adenosine monophosphate (cAMP) / protein kinase A (PKA) pathway is a highly compartmentalized signaling system. This ubiquitous pathway can have vastly different cellular outcomes depending on when and where it is turned on within a cell. Mitochondria are an excellent model of compartmentalization in cells as the mitochondrion is composed of three distinct compartments: the outer mitochondrial membrane (OMM), intermembrane space, and mitochondrial matrix (MM). Evidence supports that cAMP/PKA exists in all three of the major mitochondrial compartments and that the roles of this signaling pathway in each compartment have both shared, and distinct roles including regulation of oxidative phosphorylation(OXPHOS), steroidogenesis, and cell death/survival. However, there is controversy over which pools of PKA are responsible for control of OXPHOS. There are no tools currently available to selectively activate cAMP/PKA within specific mitochondrial compartments. Therefore, we have generated OMM and MM targeted fluorescent fusions of a small light activated adenylate cyclase (bPAC) to probe the roles and functions of OMM and MM cAMP/PKA in OXPHOS. Using microscopy, cell biology, and biochemical methods we show that PKA activity can be modulated by light in cells expressing OMM or MM‐bPAC and that the activity is confined to the OMM and MM respectively.
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
pH‐dependent studies on the reductive half‐reaction of E. coli methylenetetrahydrofolate reductase (769.13)
Authors: Schlasner Katherine , Wang Jingjing
Published: June 2021

Methylenetetrahydrofolate reductase (MTHFR) is a flavoprotein that utilizes two substrates, NADH and 5,10‐methylenetetrahydrofolate (CH2‐H4folate), in a ping pong bi‐bi mechanism. In the reaction, bound FAD is reduced by NADH and then oxidized by CH2‐H4folate. The pH dependence of the reductive half‐reaction was studied with steady‐state kinetics using the NADH‐menadione oxidoreductase assay. In this assay the reductive half‐reaction is rate‐limiting, and menadione is used to reoxidize the flavin. The wild‐type MTHFR reaction was found to be pH‐dependent with an inflection point (pKa) at 4.9. We have hypothesized that this pKa belongs to the active‐site amino acid glutamate 28 (Glu28). When Glu28 is deprotonated, the negatively‐charged side chain can stabilize the positively‐charged nicotinamide ring of NADH during the reductive half‐reaction. To determine whether Glu28 is responsible for the pKa of 4.9, we have examined a mutant enzyme with Glu28 replaced by a glutamine. Kinetic parameters were obtained for the NADH‐menadione reaction catalyzed by the Glu28Gln mutant at pH values 4.5 to 8. Our results suggest that a pKa ~5 still exists in the pH rate profile of the Glu28Gln mutant. However, instability and denaturation have plagued our studies. In current work, we are testing alternate buffers conditions to stabilize the mutant enzyme.
Interrogating the function of a putative carbon monoxide oxidase in Caulobacter crescentus (769.17)
Authors: Larkin Isaac , Crosson Sean , Colman Albert
Published: June 2021

Aerobic carbon monoxide oxidase (COX) is an oxygen‐stable enzyme that enables some bacteria to use carbon monoxide as an energy and a carbon source (1), and has the potential to contribute to the conversion of syngas, the mixture of CO, H2 and CO2 that is the product of organic waste gasification, into useful organic carbon (2). However, bacteria with COX genes tend to be extremophiles or grow slowly (Td >3 h), and thus are not ideal vehicles for biotechnological innovation (3, 4). We investigated whether CCNA_00021 (21) and 00022 (22), two genes in the fast growing and genetically tractable bacterium C. crescentus, code for a functional carbon monoxide oxidase (COX). We analyzed transcriptional data from C. crescentus to determine that 21 and 22 are upregulated during carbon starvation. To determine whether C. crescentus can consume CO as an energy or carbon source, we performed growth curves of wild type C. crescentus in rich (PYE) and Glucose Exhaustion Media in gas‐tight vessels containing different mixtures of air and CO, and then used gas chromatography to analyze the evolution of the vessels’ headspace gas. Our results indicate that while C. crescentus is more tolerant of CO than E. coli, it does not consume CO while growing in either PYE or Glucose Exhaustion Media. We therefore conclude that CCNA_00021 and 00022, though structurally similar to a COX, have a different function.
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