bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating the regulation of DNA replication origin licensing during cellular quiescence
Authors: Coleman Kate Elizabeth , Yan Feng , Major Ben , Cook Jeanette Gowen
Published: June 2021

DNA replication origins are licensed by loading of the minichromosome maintenance (MCM) complex during G1. This licensing step is inhibited during cellular quiescence (also termed G0). Licensing inhibition during G0 is an important contributor to restraining cell proliferation, but is still poorly understood. Our lab discovered that acute activation of stress MAP kinases inhibits licensing, and importantly these kinases are normally active during quiescence. We therefore hypothesize that the stress MAPKs contribute to inhibiting licensing during G0 to maintain the non‐proliferative state. To test this model, we are determining whether inhibiting stress MAP activity during G0 promotes inappropriate licensing activity and inefficient quiescence maintenance. We also considered the possibility that licensing activity during G0 is controlled by novel MCM‐interacting proteins that could be related to stress MAPK‐mediated inhibition or may work independently of MAPK activity. Thus, in an unbiased approach, we are using mass spectrometric analysis of MCM interacting proteins to identify proteins that differentially interact with G0 vs. G1 MCM complexes. Results from these combined studies will provide further mechanistic insight into quiescence control, and how perturbations in licensing activity can affect proper cell cycle exit and re‐entry. (NIH GM083024 to J.G.C and AHA12PRE10900001 to K.C.)
Investigation of the pre‐sensor 1 hairpin in minichromosome maintenance proteins 2–7.
Authors: Lam Simon Kai Wing , Sing Tina , Brandl Christopher J. , Davey Megan J.
Published: June 2021

The minichromosome maintenance proteins 2–7 (Mcm2–7) assemble into the replicative helicase in eukaryotes. To understand how this ring‐shaped helicase translocates along DNA to unwind the duplex nucleic acid, we have analyzed the structure/function relationships of the S. cerevisiae proteins. A pre‐sensor 1 (PS1) hairpin found in archaeal MCMs is thought to be involved in DNA translocation, with a conserved lysine residue predicted to contact the phosphate backbone. We mutated the analogous lysines to alanines in the putative PS1 hairpins of each of the yeast Mcm2–7 subunits. Mutation of the Mcm3 hairpin results in a loss of cellular viability. Its overexpression in the context of a wild‐type allele results in enlarged cells and a reduced growth rate. Purified Mcm2–7 complex containing the Mcm3 hairpin mutant does not unwind DNA in vitro, yet retains wild‐type levels of ATPase activity. Interestingly, individual mutation of the PS1 hairpins of the other subunits does not affect viability. However, these strains do have growth‐related sensitivity to genotoxic stress agents, and cold. Collectively, our data indicates the importance of the PS1 hairpin for Mcm2–7 function.

Support from the CIHR
Probing the nucleic acid binding modes of the single‐stranded DNA binding protein of the bacteriophage T4 replication complex
Authors: Jose Davis , Weitzel Steven E. , von Hippel Peter H.
Published: June 2021

Combining biophysical measurements on the function and control of T4 bacteriophage replication complexes with detailed structural information can throw light onto the mechanism of action of these â€̃macromolecular machinesâ€™. In this study we use the low energy circular dichroism and fluorescent properties of site‐specifically introduced fluorescent base analogues to monitor the binding of gene 32 protein (gp32), the ssDNA binding protein of the T4 DNA replication complex, to ssDNA oligomers ranging from 8 to 60 nts in length. We show that gp32 binds preferentially to the 3â€™‐end of a short ssDNA oligomer and that only two or three nts at the 3â€™ end are directly involved in the binding interaction. Loss of exciton coupling in the CD spectrum and an increase in fluorescence intensity shows that gp32 binding causes extension of sugar‐phosphate backbone of the oligonucleotide. Further, our results suggest that sliding (or â€̃shufflingâ€™) of the oligonucleotide along the nucleic acid binding cleft of the protein is not observed in the isolated single‐site gp32 DNA binding mode. Mechanisms by which gp32 binds to and covers its ssDNA targets in both an isolated and a cooperative binding mode will be discussed.
Structure‐functional study of proteins that regulate cell proliferation and differentiation
Authors: Zhu Guang , Zhou Bo
Published: June 2021

Organ development requires precise regulation of both the total number and the different types of cells. It has been shown that these two processes are linked based on mutual regulation between the cell cycle‐regulator Geminin and homeodomain ‐containing transcription factors Hox. To understand the respective molecular mechanism, we conducted structure‐functional study of Cdt1, Geminin, Mcm6 and Hox proteins by NMR and carried out relevant biochemical studies to delineate the structural basis of this regulation (Liu et al (2012) NAR 40, 3208; Zhou et al (2012) PNAS 109, 8931). We also studied histone H4–K20 mono‐methyltransferase, SET8 and its roles in cell‐cycle (Yin et al (2008) Cell Cycle 7,1423). Our results indicate that SET8 is a new cell‐cycle regulator. These works are supported from Research Grants Council of Hong Kong (RGC664109, AoE/M‐06/08, RGC663911, TUYF).
POSTMORTEM TIME AFFECTS THE INTEGRITY OF DNA EXRACTED FROM THE BRAIN AND KIDNEY OF MALE ALBINO SPRAGUE DAWLEY RATS
Authors: EBUEHI ALBERT O , BALOGUN Ayooluwa , Adenike FOWORA , EBUEHI Olufunke , AMODE Tolani , OJIEH Godwin
Published: June 2021

When an organism dies, internal nucleases existing within the cells are expected to cause DNA to degrade into smaller fragments over time that will eventually lead to alteration in the profile of the genomic structure of the organism. This study to evaluate the effect of post‐mortem time on the integrity of DNA extracted from the brain and kidney of male Sprague Dawley albino rats.

Deoxy ribonucleic acid (DNA) was extracted at different post‐mortem times (0, 24, 48 hours), from the brain and kidney of the male albino rats sacrificed via cervical dislocation. Random amplification of polymorphic DNA Polymerase chain reaction (RAPD‐PCR) was used to detect the effect of post‐mortem time on the integrity of DNA by creating the current profile of the DNA at each post‐mortem time.

The post‐mortem DNA profile observed in this study reveals that DNA degradation is a sequential time dependent process.

There is a relationship between degradation rate of nuclear DNA and post‐mortem time in the studied organs. The rat brain showed a slower degradation rate and is therefore considered as a valuable organ for studying DNA in longer post‐mortem time. The finding from this study is very useful in forensic science and investigation.
The ATPase Cycle of the Tail‐Anchored Protein Chaperone Get3
Authors: Rome Michael , Shan Shu‐ou
Published: June 2021

The cytosolic ATPase Get3/TRC40 mediates targeting of tail‐anchored (TA) membrane proteins to the endoplasmic reticulum (ER). Get3 functions as a molecular chaperone by binding to transmembrane domains of newly synthesized TA proteins and delivering them to a membrane docking complex in the ER. Previous work have shown that ATP binding and hydrolysis can regulate Get3s function and are essential for the targeting of TA proteins. However, the precise mechanisms by which this is accomplished remain unclear. Using a combination of mechanistic enzymology and biophysical methods, we delineate Get3s ATPase cycle and show that it is required at multiple stages during TA protein targeting. Our data complements previous structural studies and contributes to our understanding of how Get3 harnesses the energy of ATP hydrolysis to spatially and temporally coordinate the delivery of TA proteins to the ER membrane.
Role of the Recombination Enhancer in mating‐type switching in budding yeast
Authors: Mehta Anuja , Haber James
Published: June 2021

DNA double‐strand breaks (DSB) can be repaired by two major processes: nonhomologous end‐joining and homologous recombination. In budding yeast, repair of DSB repair occurs by gene conversion (GC) if both ends of the DSB share homology with another DNA sequence. The Saccharomyces cerevisiae mating‐type switching includes formation of a controlled DSB at the MAT locus and repair from one of two donors, HML and HMR, through the GC repair mechanism. Donor preference has been shown to be highly biased so that MATa cells recombine preferentially with HMLα and MATα cells with HMRa. This bias is determined by a ~700 bp, cis‐acting region called the Recombination Enhancer (RE). Preliminary experiments show that in the absence of RE, initiation of DNA synthesis and subsequent repair of the break is strikingly delayed. We propose that the RE region plays an important role in regulating kinetics of MAT switching.
Disruption of de Novo Mitochondrial Thymidylate Synthesis Increases mtDNA Content
Authors: Alonzo Judith Raquel , Stover Patrick J.
Published: June 2021

Mitochondrial DNA (mtDNA) synthesis is necessary for the replication and repair of damaged DNA; maintaining the proper balance of nucleotides is required to avoid mtDNA depletion and mutation. Mitochondrial deoxynucleotide triphosphates (dNTPs) are obtained from either salvage pathways or de novo biosynthesis within the mitochondria. In the mitochondria, de novo dTMP biosynthesis requires the enzymes serine hydroxymethyltransferase 2 (SHMT2), thymidylate synthase (TYMS), and dihydrofolate reductase‐like 1 (DHFRL1). In the nucleus, depletion of dTMP synthesis leads to misincorporation of deoxyuridine into nuclear DNA (nuDNA) resulting in genomic instability. SHMT2 in the mitochondria is responsible for transferring a one‐carbon unit from serine to tetrahydrofolate (THF), producing 5,10‐methyleneTHF and glycine in a reversible reaction; 5–10‐methyleneTHF is utilized by TYMS to create dTMP from dUMP.

Our goal is to determine how mtDNA content is affected by disruptions of de novo mitochondrial dTMP synthesis, using two different approaches to decrease SHMT2 activity. First, SHMT2 expression in mammalian HeLa cells was decreased using siRNA technology. Second, HeLa cells were cultured in glycine concentrations ranging from 0mM to 10mM, which inhibits the SHMT2‐catalyzed synthesis of 5,10‐methyleneTHF. The mtDNA and nuDNA copy numbers were determined by qPCR using primers specific for the mitochondrial tRNALeu(UUR) gene and a single copy nuclear gene, β2‐microglobulin. Effects of SHMT2 inhibition on deoxyuridine misincorporation into mtDNA are being assessed.

Our preliminary results suggest that mitochondrial de novo dTMP synthesis is affected by SHMT2 expression and glycine concentration. Knockdown of SHMT2 results in an increase (2.8 fold) in mtDNA copy number. Similarly, mtDNA copy number increases with increasing glycine concentration in culture media (4 fold). Supported by NIH R37DK58144.
Functional interplay of histone H1 phosphorylation and methylation
Authors: Juan Li‐Jung
Published: June 2021

Functions of histone H1 modifications are largely unknown. Our mass spectrometry analysis reveals many novel H1 modification sites, some of which are cancer cell‐specific or deregulated by oncoproteins E6 or E7 of the human papillomavirus. We identified SET7 (also named SET7/9) as a novel histone methyltransferase for H1 at K33 in vitro and in vivo. This methylation can be enhanced by E6 or E7. Moreover, we found that H1 can also be phosphorylated at S35 and it occurs exclusively at mitosis. Interestingly, H1 phosphorylated at S35 is excluded from the condensed chromatin. Further investigation shows that PKA is the major kinase for mitotic H1S35 phosphorylation. Inhibition of PKA activity altered compactness of mitotic chromatin, implying that PKA‐mediated H1S35 phosphorylation during mitosis could be involved in mitotic compaction of chromatin. In addition, a crosstalk between K33 methylation and S35 phosphorylation was observed, suggesting a functional link between these two H1 PTMs.
Probing the histone binding requirements of the Hif1 histone chaperone, a component of the Hat1 chromatin assembly complex
Authors: Szabo Stephen , Dutnall Robert N.
Published: June 2021

Hif1 (Hat1‐interacting factor 1) is a component of the Hat1 chromatin assembly complex. This complex contains the Hat1 histone acetyltransferase enzyme, that specifically acetylates histone H4, and acts in a pathway that deposits newly synthesized histone H3 and histone H4 onto DNA during chromatin assembly. The Hat1 complex is highly conserved, found in organisms ranging from yeast to humans, and plays a role in gene regulation and DNA repair. Hif1 binds histones and has histone chaperone activity. Evidence suggests that the Hat1 complex acts in histone deposition upstream of the Asf1 histone chaperone. Asf1 interacts with a heterodimer of histones H3 and H4 and blocks formation of the heterotetrameric form of histones H3 and H4 found in chromatin. This raises the question of whether Hif1 binds to a similar H3–H4 heterodimer or to a heterotetrameric complex. This is especially intriguing as Hif1 forms oligomers. To test this question, we have created site‐directed mutants of histone H3 and produced mutant H3–H4 complexes in bacteria via co‐expression methods. Mutations were introduced at the H3–H3 interaction surface that destabilize heterotetramer formation and produce obligate heterodimers. We are currently analyzing the ability of Hif1 to interact with native and mutant histone complexes to assess whether it prefers to bind heterodimeric or heterotetrameric histone complexes.
Cisplatin‐induced apoptosis in HeLa cells to determine potential mechanisms for drug resistance
Authors: Shipley Mackenzie M. , Piefer Andrew J.
Published: June 2021

The purpose of this project is to study the cisplatin‐induced apoptosis mechanism in HeLa cells. Apoptosis or programmed cell death occurs when molecular lesions are formed in genomic DNA. By using small‐interfering RNA (siRNA), the oncogene c‐Abl will be targeted and knocked down to suppress protein synthesis. c‐Abl encodes a cytoplasmic and nuclear protein tyrosine kinase. This oncogene gets translocated to the mitochondrial membrane and when DNA damage occurs, cytochrome c is released into the cytosol where eventual intrinsic apoptosis occurs. A colorimetric assay kit will be used to measure the amount of caspase‐3 present in cells before and after knockdown of c‐Abl. Caspase‐3 is a cysteine protease with specificity for aspartate residues. All caspases play an important role in the execution‐phase of intrinsic apoptosis. Trypan Blue will also be used to measure the total amount of cell death before and after knockdown of the gene target. siRNA knockdown of c‐Abl should result in decreased caspase‐3 and increased cell survival. The results of this research will determine if c‐Abl is involved in cisplatin mediated apoptosis in a cervical cancer cell line and may reveal potential drug resistance mechanisms. This research is funded by the Hartwick College Department of Chemistry.
Bortezomib Inhibits Expression of Immunosuppressive Cytokines IL‐10 and TGFβ in Cutaneous T Cell Lymphoma
Authors: Chang Tzu‐Pei , Sanacora Shannon , Manna Subrata , Singha Bipradeb , Vancurova Ivana
Published: June 2021

Cutaneous T Cell Lymphoma (CTCL) is characterized by a clonal expansion of helper T cells that release the immunosuppressive cytokines IL‐10 and TGFβ, thus suppressing the immune responses. Proteasome inhibition by bortezomib (BZ) is a promising therapeutic strategy in a number of hematological malignancies, including CTCL. Previous studies have shown that in CTCL, BZ induces apoptosis by inhibiting the constitutive activity of NFκB and expression of NFκB‐dependent anti‐apoptotic genes. In addition to the anti‐apoptotic genes, NFκB regulates transcription of immunosuppressive genes, such as IL‐10 and TGFβ. However, their regulation by NFκB is much less understood. In this study, we investigated the regulation of IL‐10 and TGFβ expression by BZ in the CTCL Hut‐78 cells. We show that BZ inhibits both IL‐10 and TGFβ expression in Hut‐78 cells. Chromatin immunoprecipitation analysis revealed that BZ inhibits NFκB p65/p50 recruitment to the promoter regions of IL‐10 and TGFβ genes. Since a variety of human malignancies utilize IL‐10 and TGFβ to evade the immune surveillance and facilitate tumor growth, a better understanding of the mechanisms how NFκB and BZ regulate transcription of these immunosuppressive genes may lead to more effective cancer therapies.

Funded by:

NIH grants GM079581 and AI085497 to I.V.
Inhibition of GSK‐3 activates CREB transcriptional targets during induction of cardiomyocyte hypertrophy
Authors: Tullai John W. , Sepulveda Sean , Brennan James F. , Moss Mary E. , Naya Frank J. , Cooper Geoffrey M.
Published: June 2021

Glycogen synthase kinase 3 (GSK‐3) is strongly anti‐hypertrophic in heart, but is inhibited by growth factors and hypertrophic stimuli. Despite its known role as a central regulator of cardiac hypertrophy, the full transcription network downstream of GSK‐3 is poorly understood. We used a systems‐level approach to delineate the transcription program downstream of GSK‐3 in neonatal rat ventricular myocytes upon the induction of hypertrophy. We first identified a set of phenylephrine (PE) induced genes, and found that 14 of the PE‐induced genes were up‐regulated by direct inhibition of GSK‐3 alone. Computational analysis revealed that CREB binding sites were statistically over‐represented for the GSK‐3 regulated genes. CREB binds to the upstream sequences of 8 of the 12 genes tested by chromatin immunoprecipitation. Upon inhibition of GSK‐3, CBP was recruited to 6 of the eight genes that had CREB present. Direct inhibition of GSK‐3 also resulted in the phosphorylation of CREB on Ser 133, suggesting a role for GSK‐3 in CREB activation. Inhibition of GSK‐3 alone induced hypertrophy marker genes at 24 hours, but did not result in an increase in protein synthesis by 48 hours. Thus, inhibiting GSK‐3 alone is sufficient to induce a partial phenotype by inducing late gene changes characteristic of hypertrophy, but not to the full hypertrophic phenotype. Supported by American Heart Association Grant 0835284N (J.W.T.).
Characterization of the interaction between Rrn3 and rpa43:Identification of a peptide that inhibits rDNA transcription and cell growth
Authors: Rothblum Lawrence , Penrod Yvonne , Rothblum Katrina
Published: June 2021

Rrn3 functions to bridge core RNA polymerase I with the preinitiation complex assembled on the rDNA promoter. One of the polymerase subunits involved in this interaction is rpa43. We have studied the interaction between rpa43 and Rrn3. We identified a 22 amino acid long domain within rpa43 is both necessary and sufficient to mediate the interaction between rpa43 and Rrn3.

We hypothesized that the addition of excess rpa43 or the 22 amino acid peptide would inhibit rDNA transcription in vitro. We added purified wild‐type or mutated (randomized 22mer) rpa43 to in vitro transcription reactions. The presence of excess rpa43 but not rpa43/random inhibited rDNA transcription. Similar results were obtained when we added either the 22mer or the randomized 22mer. The peptides were coupled to TAT peptide for transduction into cells. The random peptide had no effect on cell growth or rDNA transcription. When the wild type peptide was transduced into normal diploid human cells, it arrested cell growth. However, when introduced into cultured cancer cells, it induced cell death. In some instances we have observed 90% cell death within 8 hr. The 22mer is a small molecule inhibitor of rDNA transcription that is based on our understanding of the mechanism of rDNA transcription and appears to be specific for the rDNA transcription apparatus. We believe that it demonstrates a novel pharmaceutical target for the treatment of cancer cells.
Isolation and Characterization of the TATA binding protein (TBP) of Taenia solium
Authors: Rodríguez‐Lima Oscar , Jiménez Lucía , Zarain‐Herzberg Angel , Landa Abraham
Published: June 2021

Taeniosis/cysticercosis are diseases caused by Taenia solium and mainly affects developing countries. Little it is known about the transcriptional system in cestodes, therefore we want study and found the differences with the human and pig (host) transcription system.

The aim of this study was the identification of the elements present in the core promoter that regulates the formation of the pre initiation complex (PIC). We analyze the core promoter of different genes and identify (in silico) the cis‐elements presents. Moreover, we have cloned and characterized a TBP in T. solium. TBP is an important transcription factor to form the PIC. Besides, we identified the gene that encodes this TBP. The gen shows the presence of 4 introns in accordance with Chambon's rules and the recognition sites for splicing machinery. The amino acid sequence translated from cDNA reveal a 238‐residues protein with an expected weight of 26.7 kDa. Southern and northern blot analysis suggests the presence of one gene and two mRNA for TBP in T. solium. EMSA and supershift show a specific interaction between the TATA box of different promoters and nuclear extracts from T. solium. Our results indicate that we have isolated the gen encode a TBP and showed it interaction with TATA box from different genes.

This work was supported by grant IN219711 and CONACYT 80134 to A.L., and IN204410 to A.Z.‐H.; O.R.‐L. is supported by a scholarship from CONACyT 240037.
Enhancement of miR‐182 expression by Sp1 promotes lung cancer cell progression
Authors: Hung Jan‐Jong
Published: June 2021

Sp1 as a transcriptional factor regulates its target genes involved in cell growth, differentiation and apoptosis. Our previous study indicates that Sp1 accumulation is critical for lung tumorigenesis. Several microRNAs are also reported to be involved in cancer formation. Is there any relationship between Sp1 and mirRNA in lung tumorigenesis ? To address this issue, at first, we used the bioinformatics analysis to screen upstream flanking sequences of all intergenic miRNAs, and found that miR‐182 might be regulated by Sp1. Further study found that Sp1 could specific bind to promoter region of miR‐182 to regulate miR‐182 expression. Moreover, there is a well correlation between Sp1 level and miR‐182 level in lung cancer clinical specimens. By using three different databases of miRNA prediction screen the miR‐182 target genes. There are 161 genes were predicted to be regulated by miR‐182 possibly. Pathway analysis indicated that those genes major participated in regulation of cellular growth and proliferation. Overexpression of Sp1 decreased FOXO3 expression through miR‐182 regulation. Moreover, miR‐182 depletion delays mitotic transition by dysregulation of FOXO3 and inhibited lung cancer cells growth and tumor formation. Our data provide a mechanism of Sp1 regulated lung cancer progression through miR‐182 upregulation, thereby highlighting the importance of miRNAs biosynthesis in cancer.
Comparative analysis of transcriptional regulation by Ascl1 and Neurog2
Authors: Uhler Michael , Redmond Tanya , Huang Holly , Thompson Robert , Turner David
Published: June 2021

The proneural basic‐helix‐loop‐helix (bHLH) transcription factors Ascl1 and Neurog2 direct the differentiation of specific populations of neurons by regulating the transcription of specific sets of genes during embryonic development. Here we describe the generation of stable P19 embryonic carcinoma cells expressing comparable levels of Ascl1 and Neurog2 mRNA in a tetracycline inducible system and the resulting neuronal differentiation of these cell lines in response to doxycycline.

Microarray analysis of these cell lines showed that Ascl1 and Neurog2 overlap in their gene regulation but each bHLH protein also regulated distinct genes. We demonstrated that DNA methylation accompanied repression of the Oct4 gene by both Ascl1 and Neurog2, although DNA demethylation was not observed for any genes induced by either Ascl1 or Neurog2. While the ID1 gene showed preferential induction by Ascl1 over Neurog2, the induction of ID1 reporter constructs was dependent on distinct sets of E‐boxes within the ID1 proximal promoter. Finally, the induction of the Ppp1r14a gene was specifically induced by Neurog2 and this induction was at least partially due to preferences of Neurog2 for distinct canonical E‐box sequences within the Ppp1r14a promoter.

Together, these results suggest that further complexities in the mechanisms by which Ascl1 and Neurog2 regulate gene expression during neuronal differentiation.
Differential expression of MMP‐2 and annexin V in normal and neoplastic mouse bone cells in response to direct expression of angiotensin II
Authors: Bosch Kaitlyn , Cain Margo , Seigneur Chad , Chernin Mitch
Published: June 2021

Recent evidence has shown that Angiotensin II (Ang II) can stimulate growth in a number of different cell lines. We looked at the expression of MMP‐2, annexin V, and the Ang II receptor, AT1, to determine if these proteins are differentially regulated in normal (MC3T3) and cancerous (K7MZ) mouse bone cells. MMP‐2 has been shown to increase inflammation and metastasis, whereas annexin V has been linked to apoptosis. In order to assess whether the differences in MMP‐2 and annexin V expression were mediated by AT1, and to confirm that the Ang II produced in these cells acts via an intracrine pathway, we measured AT1 by Western blot analysis. In MC3T3 cells there was little change in MMP‐2 expression between control and the Ang II expressing cells. K7MZ mouse osteoblasts showed an increase in MMP‐2 expression as compared to the MC3T3 cells as well as an increase in expression between control and experimental (Ang II expressing) within the cell line. Cancer cells down regulate genes responsible for inducing apoptosis. Our data are consistent with this model as the K7MZ cells showed a marked decrease in annexin V expression in cells expressing angiotensin II directly. AT1 expression was not affected in either cell line by direct expression of Ang II. These data suggest that Ang II may play a role in the growth of cancer cells, and their ability to metastasize and avoid apoptosis.
Functional variants of MARCO modulate susceptibility to respiratory syncytial virus (RSV)
Authors: Marzec Jacqui M , Ciencewicki Jon M , Kleeberger Steven R
Published: June 2021

MARCO (macrophage associated receptor of collagenous structure) is a scavenger receptor that recognizes specific pathogens such as endotoxin and viral particles. We previously identified MARCO variants that associate with susceptibility to RSV disease, however the mechanism is unclear. We sought to identify functional variations in MARCO that modulate pathogenesis of RSV‐induced lower respiratory tract disease. The MARCO promoter was sequenced and aligned to identify variant single nucleotide polymorphisms (SNPs). Deletion constructs and MARCO polymorphic variants were amplified from genomic DNA and cloned into a pGL3 vector, transfected into A549 and Beas2B cells, and basal and RSV‐inducible MARCO activity assessed. We used gel shift analysis for binding efficiency and confirmation of transcription factor binding sites. One novel SNP within the MARCO promoter associated with RSV susceptibility in vitro, and another was located in a putative ARE site. We found a significant reduction in baseline transcriptional activity with the ‐ 1118A allele and the ‐156G allele. Similar results were found for transiently transfected cells exposed to RSV. Overall binding efficiency was reduced with the ‐156C allele, however NRF2:MARCO binding was enhanced. We conclude that functional SNPs in MARCO modulate RSV susceptibility.

Support: NIEHS Division of Intramural Research.
Gene expression of (pro)renin receptor is downpregulated in heart of transgenic mice with acute myocardial infarction
Authors: Ribeiro Amanda Aparecida , Amorin Rebeca Padrão , Fernandes Maria José Silva , Irigoyen Maria Cláudia , Pesquero Jorge Luiz , Casarini Dulce Elena
Published: June 2021

In this study we examined the expression of (pro)renin receptor (PRR) and renin in hearts of transgenic mice that express rat tonin with acute myocardial infarction (AMI). We used male 10 to 12 week‐old wild type controls (CT, C57 black six) and mice expressing rat tonin (TGM(rTon)). All animal were genotyped and divided into 2 subgroups: TGM(rTon) Sham and TGM(rTon) AMI; CT Sham and CT AMI. Expression levels of RPR and renin mRNAs were determined using quantitative RT‐PCR. In TGM(rTon) AMI, RPR mRNA were significantly decreased in all structures compared to CT AMI (AT:0.52±0.009 vs. 1.02±0.119; LV:0.108±0.036 vs. 1.044±0,160 and RV:0.523±0.05 vs. 0.613±0.146, respectively). The TGM(rTon) AIM showed significantly decreased RPR mRNA levels in the LV compared to TGM(rTon) Sham (0.571±0.081 vs. 1.067±0.117, respectively). In TGM(rTon) AIM, renin mRNA levels was significantly lower for all structures analyzed compared to CT AMI (AT: 0.012±0.005 vs.1.103±0.295; LV: 0.063±0,03 vs. 0.558±0,206; RV: 0.055±0,007 vs. 1.670±0.56, respectively. In TGM(rTon) AIM, renin mRNA levels was significantly lower for AT and RV compared to TGM(rTon) Sham (AT: 0,153 ± 0,01 vs. 0,277±0,018, VD: 0,194±0,025 vs. 0,523±0,05, respectively. The results suggest that tonin can influenciate in the synthesis of RPR and renin mRNA leves in the heart. Supported by Fapesp (10/19989–4).
Understanding the basis of pathogenicity in the R592W mutant of human mitochondrial alanyl‐tRNA synthetase
Authors: Borchardt Jennifer , France Katherine , Chihade Joseph
Published: June 2021

We are exploring the effects of recently discovered (Götz, et al., (2011) Am. J. Hum. Genet. 88, 635) pathogenic mutations in human mitochondrial alanyl‐tRNA synthetase (mt AlaRS) on tRNA recognition and enzymatic function. These authors found an R592W mutation in multiple patients with severe infantile cardiomyopathy. R592 lies within the editing domain of mt AlaRS, but is distal to the aminoacyl editing active site. To characterize the editing function of mt AlaRS, we have constructed an editing‐defective C719A/Q622H mutant of the enzyme. While this editing defective mutant effectively mischarges human mt tRNAAla with serine, no accumulation of Ser‐tRNAAla is observed with wildtype or R592W mt AlaRS. We are currently investigating the hypothesis that, rather than disabling the enzyme's ability to hydrolyze misacylated tRNAs, the R592W mutation enables hydrolysis of the correctly acylated Ala‐tRNAAla product in the editing site of mt AlaRS.

This work was supported by Carleton College and the Howard Hughes Medical Institute.
Changes in synapsin 1 phosphorylation and tubulin acetylation in mice deficient in protein L‐isoaspartyl methyltransferase
Authors: Qin Zhenxia , Aswad Dana W.
Published: June 2021

Protein L‐isoaspartyl methyltransferase (PIMT) repairs damaged proteins that contain abnormal isoaspartyl (isoAsp) peptide bonds. PIMT knockout (KO) mice accumulate high levels of isoAsp damage in the brain and succumb to fatal epileptic seizures at 28–60 days after birth. Synapsin 1 and tubulin are major endogenous substrates for PIMT in brain, which could help explain the abnormal synaptic transmission and disorganized microtubules seen in the KO mice. To evaluate the effect of PIMT deficiency on the function of these proteins in vivo, we used Western blotting with modification‐specific antibodies to assess the state of phosphorylation of synapsin 1 and acetylation of tubulin. In female mice, phosphorylation of synapsin 1 at the Ser‐9 (protein kinase A) site was increased 145% in KO versus WT (wild type) mice. In males the increase was 22%. There was no change in phosphorylation at the Ser‐603 (CaM kinase II) site in either male or female mice. Acetylation of α‐tubulin at Lys‐40 was decreased approximately 28% in both male and female KO mice. These results show that isoAsp accumulations in synapsin 1 and tubulin are associated with functional changes in these proteins in vivo. We propose that these changes contribute to the aberrant neurotransmission and abnormal microtubule organization caused by PIMT deficiency.
Degradation of the transcription factor Nrf2 in promyelocytic leukemia‐nuclear bodies
Authors: McIntosh Deneshia , Arinze Ifeanyi
Published: June 2021

The cellular abundance of the redox‐sensitive transcription factor Nrf2 is controlled by Keap1, an inhibitory protein that functions as a substrate adaptor for ubiquitylation of Nrf2 by the Cullin3/RING box1 (Cul3‐Rbx1) E3 ubiquitin ligase complex, thereby targeting Nrf2 for proteasome‐dependent degradation in the cytoplasm. Nrf2 can also be degraded in the nucleus but mechanistic details are lacking. Here, we show that promyelocytic leukemia‐nuclear body (PML‐NB)‐enriched preparations of HepG2 cells treated with arsenic trioxide (ATO) contained modified (sumoylated) Nrf2 and the poly‐SUMO‐specific ubiquitin E3 ligase RNF4, but not Keap1. Overexpressing RNF4 decreased the steady‐state levels of Nrf2 whereas the catalytically inactive RNF4 did not. The proteasomal inhibitor MG‐132 interfered with this RNF4‐induced decrease. In MG‐132‐treated cells, the content of ubiquitylated Nrf2 was higher in cells transfected with wild‐type RNF4 than in cells transfected with catalytically inactive RNF4. RNF4 also decreased the half‐life of Nrf2, measured in PML‐NB‐enriched fractions prepared from ATO‐treated cells. Given that treatment with ATO enhances SUMO‐2/3 attachment to Nrf2, these data suggest that RNF4 induces poly‐ubiquitylation of poly‐sumoylated Nrf2, leading to its degradation by proteasomes in PML‐NBs.

Supported by NIH grants #5T32HL007737, 2 SD1MD000104 and SC1CA143985
Investigating the Roles of Wntless (Wls) Isoforms in Wingless (Wg) Signaling
Authors: Lombardi Nicholas J , Selva Erica
Published: June 2021

Wnts are secreted signaling molecules that have essential functions in development and adult homeostasis. Wnt family proteins and their signaling pathway are conserved from humans to invertebrates. Wingless (Wg) is the founding Drosophila Wnt family member. In the wing disc, secreted Wg forms a gradient that activates short‐ and long‐range target in a concentration dependent manner. Wntless (Wls) is a conserved multi‐pass transmembrane protein that binds to Wg and escorts it through the secretion pathway and exists in two isoforms, Wls‐A and Wls‐B. The goal of this research was to determine the relative abundance of the two Wls isoforms in wing discs and determine differences in their functions. WlsB, the predominate transcript, harbors a retromer retrieval signal necessary for Wg secretion in the wing disc, which is absent in WlsA. Expression of either Wls‐A or Wls‐B is sufficient to rescue wls‐/wls‐ flies to adulthood. Using the UAS‐Gal4 system the phenotypic consequences of overexpressing each was explored. Wls‐A was lethal when expressed uniformly and had various strong phenotypes when overexpressed in different patterns in the wing imaginal disc. Furthermore, WlsA causes a cellular redistribution of Wg in ligand producing cells. Wls‐B displayed no phenotypes when overexpressed in the same manner, but causes a minor reduction in Wg in ligand producing cells. Funding: UD undergrad research, NSF, HHMI
Role of membrane‐proximal basic residues in cell surface trafficking of GPR15
Authors: Shikano Sojin , Bernstein Joshua , Okamoto Yukari
Published: June 2021

Membrane‐proximal basic residues (MPBRs) in the cytoplasmic region are one of the important factors that determine the topology of membrane proteins during biogenesis in the endoplasmic reticulum (ER), known as a positive‐inside rule. However, MPBRs have been also found to be critically involved in the ER export of certain membrane proteins such as Golgi‐resident glycosyltransferases. Here we investigated the role of cytoplasmic MPBRs (Arg310Arg311) in the cell surface trafficking of GPR15, a G protein‐coupled receptor that serves as an HIV co‐receptor. The mutation of Arg310Arg311 to Ala did not alter the topology but abolished the O‐glycosylation and cell surface expression of GPR15. Immunocytochemistry indicated that Ala mutant can exit ER and reach ER‐Golgi intermediate compartment (ERGIC) and cis‐Golgi, but not trans‐Golgi. Our study suggests the possibility that MPBRs play a pivotal role in the anterograde trafficking of a broad range of membrane proteins.
Influence of proteasomal degradation of RNA‐binding protein dTIS11 on ARE‐mediated decay
Authors: Ngoc Long Vo , Kruys Véronique , Gueydan Cyril
Published: June 2021

dTIS11 is an RNA‐binding protein responsible for the processing of AU‐rich element (ARE)‐containing mRNAs in Drosophila melanogaster. In response to immunological challenge, coordination of dTIS11 levels with that of its target genes is crucial for the implementation of their transient expression profiles. We observed that this mechanism could operate in absence of transcriptional regulation of the tis11 gene, suggesting the presence of post‐translational regulation processes. Here, we report that dTIS11 protein stability is tightly regulated in resting drosophila S2 cells, by a process that involves proteasomal activity, phosphorylation and RNA‐binding. Upon immunological challenge, modifications to dTIS11 alter its decay rate, adjusting to the increasing levels of target ARE‐containing mRNAs. This work was supported by a doctoral grant of the Fonds pour la Recherche en Industrie et Agriculture
Variability in Fusogenic Activity of Human Metapneumovirus Fusion Protein from Different Strains
Authors: Klimyte Edita , Chang Andres , Dutch Rebecca E.
Published: June 2021

First identified in 2001, the paramyxovirus human metapneumovirus (HMPV) is a novel pathogen that causes viral respiratory disease in infants, the elderly, and immunocompromised patients worldwide and is the second most common cause of pediatric lower respiratory illness, following the closely related RSV. Despite its clinical significance, little is known about its entry pathway, complicating the search for antivirals. Of the three surface glycoproteins expressed on the viral membrane, the attachment protein, a small hydrophobic protein, and the fusion protein F, only the F protein is required for membrane fusion and infectivity. The F protein undergoes a dramatic conformational change in order to bring the viral and target cell membranes together. This conformation change in HMPV strain CAN97–83 has been shown to be triggered by low pH. However, low pH triggering varies between F proteins of different strains. In this study we characterized fusogenic activity of strains representative of all four clades using two independent fusion assays, syncytia formation and luciferase reporter gene assay. Our results suggest that both the overall fusion level and the amount of stimulation by low pH vary between F proteins of different strains. Comparison of F protein sequence will be used to identify potential critical residues. This research was supported by NIH grant R01AI051517.
Quantitative Proteomics to Profile Post‐translational Modifications During M Phase: Interplay Between O‐GlcNAcylation and Phosphorylation
Authors: Whelan Stephen A. , Tan Ee Phie , Costello Catherine E. , McComb Mark E. , Slawson Chad
Published: June 2021

During mitosis, proteins undergo a variety of post‐translational modifications (PTM) in order to alter function and localization. Our objective is to determine the interplay between O‐GlcNAcylation and phosphorylation on proteins during mitotic progression. O‐GlcNAc is the attachment of a single N‐acetylglucosamine moiety to serine and threonine residues in nuclear and cytoplasmic proteins. O‐GlcNAc transferase (OGT) adds the modification and O‐GlcNAcase (OGA) removes the modification. O‐GlcNAc is similar to phosphorylation as both modifications are responsive to the extracellular environment, nutrients, and growth hormones. Using SILAC (Stable Isotope Labeling of Amino Acids in Cell culture) labeling, we are able to quantitate differences in cells with either an OGT or OGA gain of function. Combined samples were digested and fractionated to enrich for O‐GlcNAcylated or phosphorylated peptides. We identified numerous proteins whose expression or phosphorylation status was altered by aberrant O‐GlcNAcylation. These results demonstrate the interconnectedness of these two modifications in regulating mitotic progression. Furthermore, these data suggest that diseases that disrupt one PTM will also alter the actions of the other PTM.
Rab7‐dependent microautophagy in the visceral endoderm is essential for mouse early development
Authors: Wada Yoh , Sun‐Wada Ge‐Hong
Published: June 2021

During early embryogenesis, before the conceptus forms the placenta, maternal nutrients and signaling molecules must reach the embryo proper through a tightly sealed epithelial tissue, the visceral endoderm (VE). The VE serves as a signaling center for embryogenesis, where exocytic and endocytic processes integrate signal production, perception, and termination. However, the endocytic process in this tissue has not been well characterized. We optimise three schemes for tracing the endocytosis in the VE. In utero labelling scheme, an endocytic tracer is introduced into circulation of a pregnant mother to follow endocytosis. Rodent embryos are known to internalise maternal immunoglobulins, thus steady‐state levels of endocytosis can be visualised by subcellular localization of mouse IgG. We also developed an in vitro labelling method which allows pulse‐labelling and chase experiments thus we can follow the temporal orders of events. We show that the endosomes of visceral endoderm cells are delivered via a unique microautophagy‐like process to the apical vacuole, a large compartment exhibiting lysosomal characteristics. This process requires the functions of rab7, a small GTP‐binding protein regulating the late endocytic pathway. Loss of rab7 function results in severe defects in embryonic patterning during gastrulation. Our results indicate that the microautophagic process and flow of the endocytic membrane play essential roles in early embryonic development.
Activated GTPase movement on an RNA scaffold drives cotranslational protein targeting
Authors: Shen Kuang , Arslan Sinan , Akopian David , Ha Taekjip , Shan Shu‐ou
Published: June 2021

Signal Recognition Particle (SRP) and its receptor (SR) are GTPases that translocate ribosome‐nascent chain complexes (RNCs) from cytosol to cellular membranes. SRP recognizes the RNC, and through interactions with SR, brings the RNC to the membrane. Subsequent rearrangements activate GTP hydrolysis and load the RNC onto the translocon. During this process, the SRP RNA plays an indispensible role in stimulating both SRP‐SR complex formation and the following GTP hydrolysis. Previous studies hinted that the SRP RNA is a bi‐functional molecule with two distinct functional ends, the tetraloop end and the distal end, that each stimulates the SRP‐SR complex formation and GTP hydrolysis steps. Here we used single molecule technique to directly visualize the global rearrangement of the GTPase complex from the RNA's tetraloop end to the distal end, traveling over 100 Angstrom in the targeting reaction. Moreover, we showed that the rearrangement is tightly controlled by the RNC and the translocon. This large‐scale movement of the GTPase complex provides a molecular mechanism of coupling GTPase activation to the transfer of RNC, thereby ensuring productive protein targeting.
Proteasomal complex regulation by heat‐shock proteins in cancer
Authors: Tsvetkov Peter , Taipale Mikko , Lindquist Susan
Published: June 2021

Proteasome mediated protein degradation is a major component of the cellular machinery that maintains the protein homeostasis. The disruption of the proteasomal function induces proteotoxic stress and activates the cellular heat shock response. There are two distinct proteasomal degradation pathways, the ubiquitin dependent proteasome pathway mediated by the 26S proteasomes and the ‐ubiquitin independent proteasomal degradation pathway mediated by the 20S proteasome. The cross talk between the chaperone and ubiquitin system has been highly explored and characterized, however little is known on the direct regulation of heat shock proteins on proteasomal integrity and function in mammalian cells. In this work we try to set the framework to examine the regulatory roles that Hsps might have in regulating proteasomal complex function and integrity. By using high throughput protein‐protein interaction assays and masspectrometry we examine the association of cellular proteins specifically chaperones to the proteasomal machinery following proteotoxic stresses. Our work examines the regulatory role of Hsps in maintaining protein homeostasis by directly regulating the proteasomal complex function and how this interplay contributes to cancer cell proliferation.
Title: Effect of Insulin‐like Growth Factor 1 on Cobalamin Composition in Neuronal Cells
Authors: Li Yiting , Deth Richard
Published: June 2021

Cobalamin is an essential co‐factor for methionine synthase (MS), which catalyzes the conversion of homocysteine to methionine, however, oxidization of cobalamin halts MS activity. In cultured neuronal cells reactivation of MS requires the antioxidant glutathione (GSH) to produce the active cofactor methylcobalamin (MeCbl). Hydroxocobalamin is rapidly and irreversibly converted to glutathionylcobalamin (GSCbl) in the presence of glutathione, then to MeCbl in the presence of S‐adenosylmethionine. Cysteine is the rate‐limiting precursor for GSH synthesis, and in neurons uptake via excitatory amino acid transporter 3 (EAAT3) provides the predominant source of cysteine. Insulin growth factor 1 (IGF‐1) increases cellular cysteine uptake by promoting cellular surface expression of EAAT3. We investigated the influence of IGF‐1 on cellular redox status and cobalamin composition in SH‐SY5Y human neuroblastoma cells. HPLC separation and electrochemical detection were used to measure six intracellular cobalamin species, as well as the levels of GSH and its oxidized GSSG form. We found that GSCbl is a prominent intracellular cobalamin species in SH‐SY5Y cells. IGF‐1 treatment shifted the cellular environment to a more reduced state by increasing intracellular levels of GSH and the GSH/GSSG ratio, leading to an increased level of GSCbl. This novel action of IGF‐1 allows it to regulate cellular redox status.
Substrate specificity of Rv0045c, a bacterial esterase from Mycobacterium tuberculosis
Authors: Savas Christopher Peter , Gehring Alexandra , Johnson R. Jeremy , Hoops Geoffrey
Published: June 2021

Serine hydrolases have emerged as novel therapeutic targets for treating TB infection as they play essential roles in virulence and the maintenance of latent TB infections. Herein, we characterize the substrate specificity and binding pocket structure of Rv0045c, a serine hydrolase and important metabolic regulator from M. tuberculosis. Utilizing a diverse library of fluorogenic ester substrates and comprehensive alanine scanning mutagenesis of the binding pocket and a flexible loop, we defined the substrate selectivity profile of Rv0045c and hotspots for substrate and potential inhibitor binding. Rv0045c shows highest activity toward shorter (< 6 carbons) polar esters, matching with the selectivity profile of homologous bacterial hydrolases, but retains broad specificity toward short bulky or aromatic esters. The binding pocket loop region of Rv0045c had no significant effect on its substrate specificity, but two residues surrounding the catalytic binding pocket were found to significantly increase the substrate specificity and catalytic activity of Rv0045c. The map of the binding pocket and increased activity of some of the binding pocket variants present starting points for directed evolution of novel substrate specificities. Funding provided by NSF TUES DUE‐1140526