bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of BRCA1 by SIRT2
Authors: Minten Elizabeth , Zhang Hui , Li Chunyang , Head PamelaSara , Yu David 
Published: June 2021

In our everyday lives, we are constantly being exposed to DNA damaging agents, both from internal and external sources. The most deleterious type of DNA damage a cell can suffer is the DNA double‐stranded break (DSB). Breast Cancer 1 (BRCA1) is a protein necessary for the proper repair of resultant DNA DSBs through the homologous recombination (HR) pathway. Defects in BRCA1 have been linked to different types of cancer in both men and women, including breast, ovarian, and pancreatic cancer. However, the regulation of BRCA1 is not yet well understood, and in many cases, how defects in this pathway lead to an increased risk of developing cancer is also unknown, making preventative care and treatment of resulting cancers more difficult. Our lab has discovered that SIRT2, a histone deacetylase and putative human tumor suppressor, plays a crucial role in the DNA damage response (DDR) and repair of DNA DSBs. We have shown that depletion of SIRT2 impairs HR and increases cell sensitivity to IR in a deacetylase‐dependent manner. A mass spectrometry analysis showed SIRT2 interacts with a number of proteins involved in DDR, including BRCA1. We validated the interaction between SIRT2 and BRCA1 and also found SIRT2 deacetylates BRCA1 both in vitro and in cells. Depletion of SIRT2 and subsequent deacetylation of BRCA1 decreases BRCA1 protein levels in cells and thus impairs HR. Our results show SIRT2 is a novel regulator of BRCA1 and is critical for the repair of DNA DSBs through HR. These findings provide insight into how dysregulation of SIRT2 or BRCA1 can result in genomic instability and lead to the development of cancer.Support or Funding InformationDr. David Yu, NIH R01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Link Between Mutations in MutS DNA Repair Protein and Lynch Cancer Syndrome
Authors: Kessler Emily , Liu Juan , Awad Helena , Hingorani Manju 
Published: June 2021

The Mismatch Repair (MMR) pathway is responsible for correcting errors in DNA and is thus essential for maintaining genome stability in all living organisms. Several studies have shown that mutations causing defects in MMR protein structure and function lead to hypermutation. The increased level of mutations results in serious consequences for human health, highlighted by Lynch Syndrome (LS), a hereditary predisposition to colorectal and other cancers (1). The first protein in the MMR pathway, MutS, recognizes mispaired bases or short insertion‐deletion loops (IDLs) and utilizes its ATPase activity to initiate DNA repair (2). A long‐term study in the Hingorani laboratory examines cancer‐linked single amino acid mutants of the human MutS protein (using Thermus aquaticus MutS as a model system) in order to understand how the changes affect protein structure and function, and alter MMR. This work involves kinetic analysis of MutS activities, and related dynamic conformational changes in the protein as it utilizes ATP to work on DNA. The results will enable construction of a complete mechanism of action for each mutant protein, and help us determine whether the mutants are compromised in their ability to target mismatches, and/or bind and hydrolyze ATP, and/or undergo necessary conformational changes, and therefore understand why they disrupt DNA mismatch repair and give rise to Lynch Syndrome. My project focuses on fluorescence‐based stopped flow kinetic experiments to monitor the conformational changes of wild type MutS and several LS‐linked mutants as they bind +T insertion‐containing DNA and catalyze ATP hydrolysis. In addition to generating new information on each mutant, the findings will advance the proof‐of‐principle concept that detailed analysis of the structure, dynamics, and catalytic activities of individual MutS mutants is both feasible and can reveal critical information for understanding the molecular basis of Lynch Syndrome.Support or Funding InformationNational Institutes of Health R15 GM114743; The Beckman Scholars Program, Arnold and Mabel Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Connections between the DNA damage response and mRNA processing
Authors: NAZEER FATHIMA I. , Skidders Morgan , Dean Emily , Hofler Alex , Moore Claire 
Published: June 2021

Eukaryotic cells have evolved multiple mechanisms to deal with DNA damage in order to prevent disease. In the presence of helix distorting DNA damage, RNA polymerase II (RNAPII) blockage at damage sites can lead to RNAPII degradation and activation of the DNA damage response (DDR), if the damage is not repaired promptly. Our hypothesis is that proteins involved in RNAPII degradation, Elc1 and Def1 are also involved in regulating the DDR and other related cellular processes like mRNA maturation, in response to DNA damage. Our preliminary data suggest that Def1 may be involved in activating the DNA damage response while Elc1 is involved in blocking mRNA maturation after UV‐type DNA damage. We are currently working on purifying the Yeast ElcI complex using affinity chromatography to assess if our purified Elc1 complex is able to degrade factors involved in mRNA maturation, in‐vitro. In addition, we are also constructing deletion strains to test if other protein factors involved in DNA repair play a role in blocking mRNA maturation. Taken together, our data point to an unexpected interconnection between the DNA damage response and mRNA processing pathways.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Non‐Native Structure Present in the Unfolded Ensemble May Initiate Aggregation of ALS variants of Superoxide Dismutase (SOD1)
Authors: Cohen Noah , Bilsel Osman , Matthews C. Robert 
Published: June 2021

Aggregates of Cu, Zn Superoxide Dismutase (SOD1) in the spinal cord are a hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. Mutations throughout the SOD1 sequence have been found to cause ALS, suggesting a general gain‐of‐function mechanism for the toxicity of this disease. We have previously argued that the aggregation of the unfolded chain, following synthesis on the ribosome and prior to maturation, provides a parsimonious explanation for the common phenotype. To determine whether segments of unfolded SOD1 are capable of adopting structure prior to the exceedingly slow (>10 sec) productive folding reaction, we dissected SOD1 into peptides that were labeled with a donor/acceptor FRET pair at or near their N/C‐termini. Time‐resolved FRET (tr‐FRET) analysis revealed that the peptide corresponding the C‐terminal region of SOD1 forms urea sensitive structure. Utilizing 2‐dimensional Maximum Entropy Modeling (MEM), we found that this peptide undergoes a discrete transition between a compact state and a random coil‐like structure. Mutations show this collapse results from a non‐native intramolecular interaction involving a hydrophobic sequence at the C‐terminus as well as a polar/charged sequence at the N‐terminus. Ongoing studies are examining the behavior of these segments in the full‐length protein with tr‐FRET. These results are consistent with the results of proteolysis and crystallographic studies from other labs and together suggest that this region of SOD1 may present a potential target for novel therapeutics to treat ALS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilizing homology of Wound Inducible Transcript 3.0 (WIT3) as a stepping stone to investigate the function of Suppressor of IKKepsilon (SIKE)
Authors: Dawood Mariam , Bell J. Ellis , Bell Jessica K. 
Published: June 2021

Wound inducible transcript 3.0 (WIT3.0) is a novel cytoskeleton protein that regulates fibroblast migration and initiates rapid wound closure. WIT3 shares ~50% sequence identity with an innate immune protein of unknown function, Suppressor of IKKepsilon (SIKE), that also associates with cytoskeletal proteins. We hypothesize that SIKE and WIT3 will have similar biochemical and biophysical characteristics that contribute to a shared function. To assess these similarities, we undertook characterization of WIT3 structure utilizing circular dichroism, fluorescence‐based thermal shift assays, ligand binding studies using an ANS reporter, and crosslinking with BS3. Using PHYRE2, a WIT3 model was predicted that consisted of an alpha helical coil with 30% disordered regions. The secondary structure of WIT3 was assessed via circular dichroism. Wavelength spectra is consistent with an alpha helical protein and thermal melt data showed a linear unfolding pattern with Tm of 37°C, suggesting a minimally stable protein. As part of the PHYRE2 model, 3DLigandSite proposed a zinc binding site. To assess an interaction between WIT3 and divalent cations, WIT3‐ANS titration and fluorescence‐based thermal shift assays were completed +/‐ Mg, Mn, Ca, Ni and Zn. An interaction between WIT3 and Zn was confirmed where, in the presence of zinc, WIT3's affinity for ANS was 2 fold higher than in the absence of zinc. The initial comparison suggests that WIT3 and SIKE share similar helical structure and similar FTS melt curve topography indicating exposed hydrophobic residues prior to denaturation, but different structural stability and a potential divalent cation binding site on WIT3. Together, these data suggest that WIT3 and SIKE share several biochemical characteristics that may allow these two proteins to have complimentary function.Support or Funding InformationStudies were funded in part by USD SURE.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tuberculosis serine hydrolase variable expression, isolation, and characterization under hypoxia conditions
Authors: Severinac Luke Edward , Johnson R. Jeremy 
Published: June 2021

Tuberculosis (TB), caused by infection by Mycobacterium tuberculosis, is the deadliest infectious disease worldwide. Complicating the treatment of TB is its ability to transition between an intransient dormant state and an active infectious state. One class of enzymes required for the maintenance of this dormant state and the transition into the active state is serine hydrolases. Our research goal was to globally characterize serine hydrolase activity in actively growing M. smegmatis and under hypoxia conditions that mimic dormant growth. To measure hydrolase activity, lysates were prepared under different growth conditions and then separated by native‐PAGE. Native‐PAGE gels were incubated with a library of fluorogenic ester substrates and hydrolase activity quantitated based on fluorogenic substrate activation. By following the shift in fluorogenic activation, we were able to measure changes in hydrolase protein expression versus fluorogenic substrate and in relation to induction of dormant growth conditions. Reactive hydrolases were excised from the gel and identified by mass spectrometry. Our results provided a global map of mycobacterial serine hydrolase activity and identified starting points for finding novel drug targets for dormant TB.Support or Funding InformationThis work was supported by a grant from the National Institutes of Health (NIH 1 R15 GM110641‐01A1)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression and functional characterization of active nicotinic acid dehydrogenase from Pseudomonas fluorescens Pf5
Authors: Brownstein Nathan Mark , Snider Mark J. 
Published: June 2021

Many compounds created as a byproduct of industrial processes or synthetic products are having a significant, negative affect on the environment. N‐Heteroaromatic compounds (N‐HACs), make up many of these environmental pollutants. A proposed solution to environmental damage by N‐HACs is bioremediation: using engineered microorganisms to clean areas affected by pollution. Nicotinic acid (NA) is an N‐HAC which is present in a wide range of synthetic products and is an environmental pollutant. Many microorganisms have evolved the ability to catabolize N‐HACs, including several capable of using NA as their sole source of carbon. One organism that degrades NA is Bacillus niacini. B. niacini is of interest because it degrades NA by a pathway unlike other organisms. Characterizing B. niacini’s NA degradation pathway is potentially important for accomplishing the long‐range goal of developing effective bioremediation strategies. The first step in B. niacini's putative NA catabolism pathway is through the nicotinic acid dehydrogenase NicAB, which is encoded by several ORFs in the gene cluster hypothesized to be responsible for NA degradation. Previous attempts to characterize the activity of NicAB expressed in E. coli have been unsuccessful and have demonstrated that the B subunits of NicAB are deficient in molybdenum, an essential metal for its MCD cofactor. The activity of NicAB will be characterized upon expression in Pseudomonas fluorescens Pf5, a closely related organism to the NA degrading bacteria Pseudomonas putida. P. fluorescens Pf5 may have MCD maturation proteins more compatible with NicAB. We show that P. fluorescens Pf5 has no endogenous NA dehydrogenase activity via HPLC. Future work will test whether knock‐in of the genes encoding NicAB into P. fluorescens Pf5 will show whether NA dehydrogenase hydroxylates NA and 6HNA. The activity and substrate binding of functional NA dehydrogenase will be characterized by kinetic analysis.Support or Funding InformationHenry J. Copeland Fund for Independent StudyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Insights into Peptide Recognition and Modification by the Radical SAM Enzyme SuiB
Authors: Davis Katherine M. 
Published: June 2021

Posttranslational modification of ribosomally synthesized peptides provides an elegant means for the production of biologically active molecules. Unlike nonribosomal peptides, which are assembled by large multimodular enzymes, ribosomally synthesized and posttranslationally modified peptides, or RiPPs, have comparatively simple biosynthetic pathways. Modifications to the core sequence of the precursor peptide are installed by tailoring enzymes, however, the structural basis for substrate recognition in these reactions remains unknown. Using X‐ray crystallography and computational modeling, we examine the role of the N‐terminal leader peptide in the biosynthesis of a streptide homolog, a recently identified peptide natural product with an intramolecular lysine–tryptophan crosslink installed by the radical S‐adenosylmethionine (SAM) enzyme, SuiB. We evaluate a series of structures that not only help to elucidate the formation of the C–C crosslink but also provide insights into the interplay between RiPP precursor peptides and tailoring enzymes more generally. Surprisingly, although the N‐terminal domain of SuiB adopts a typical RRE (RiPP recognition element) motif, which has been implicated in precursor peptide recognition, we observe binding of the leader peptide in the catalytic barrel. Computational simulations support a mechanism in which the leader peptide guides posttranslational modification by positioning the cross‐linking residues of the precursor peptide within the active site. Together, these findings reveal unanticipated roles for the RRE and other domains during RiPP biosynthesis by radical SAM enzymes.Support or Funding InformationThis work was supported by the Arnold O. Beckman Postdoctoral Fellowship program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis of Mono‐substituted Anthraquinone Derivatives to be Used in the Purification of Lactate Dehydrogenase
Authors: Retzlaff Brandon David , Mueller Jaime 
Published: June 2021

Cibacron Blue 3G‐A has been previously used in biochemical laboratories for purification of enzymes containing the dinucleotide fold. Lactate dehydrogenase is one such enzyme that can be purified using Cibacron Blue 3G‐A in affinity chromatography. Rising costs and inaccessibility to Cibacron Blue 3G‐A have created a demand to find a more cost effective replacement. Electron withdrawing and donating properties of the anthraquinone core substituent groups are believed to affect the affinity of the core for various enzymes. A range of anthraquinone dyes were synthesized to determine the importance of the substituents' electronic properties with the coupling of ortho substituted aniline nucleophiles to the anthraquinone core via a diazonium salt link. The aniline substituents include: methyl, methoxy, chloro, and sulfonyl groups. For each anthraquinone dye synthesized, the λmax value was determined to effectively monitor the kinetics of the dyes. Spectrometric assays measured the oxidation of NADH to NAD+ determining the dye's affinity for enzymes containing the dinucleotide fold.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Construction of Hybrid Inhibitors for Metalloproteinase Targeting
Authors: Quinto Laura B. , Berumen Gregory I. , Kehoe Haixing P. , Van Deventer James A. 
Published: June 2021

Although cancer‐promoting activities of extracellular proteases and peptidases in the tumor microenvironment are well known, the disruption of individual enzymes in this environment remains a fundamental challenge. Individual matrix metalloproteinases (MMPs), in particular, have proven difficult to selectively inhibit due to their structural similarities. To better understand the roles of these enzymes in cancer progression, and to potentially begin to target them in vivo, selective inhibitors of these enzymes are needed. We are developing a protein‐small molecule “hybrid” approach to meet this need by conjugating inhibitory small molecules to single‐chain variable antibody fragments (scFvs) that selectively bind a unique target.To this end, we have established a platform to introduce a versatile range of chemical functionalities into scFvs on the yeast surface. Using amber suppression, we can introduce noncanonical amino acids (ncAAs) at specific sites in displayed scFvs that are predicted to be close to the antigen binding site. In our proof‐of‐concept model, we have been investigating the possibility of adding enzyme active site‐targeting chemical groups to scFvs that bind to MMP‐9. Flow cytometry results show that constructs containing ncAAs with an azide side chain retain MMP‐9 binding. Importantly, introducing the azide functionality enables us to conjugate MMP‐targeting small molecules to displayed scFvs using bioorthogonal copper‐catalyzed azide‐alkyne cycloadditions (“click chemistry”). We have investigated installation of MMP‐targeting small molecules in different antibodies and at various scFv positions. In some cases, we see almost complete reaction, but the extent of reaction across antibodies and sites is dependent on the small molecule used and the site of ncAA incorporation. Systematic exploration of reaction conditions including time and click chemistry ligand has led to conditions that enhance the extent of reaction of small molecules at different sites. Work to evaluate enhancements to binding and inhibition of MMP‐9 with the resulting antibody‐small molecule “hybrid” conjugates is ongoing. We anticipate that adding more chemical functionality into proteins will facilitate selective targeting of broad classes of enzymes active in the tumor microenvironment and in other complex diseases.Support or Funding InformationTufts Summer Scholars, Tufts Faculty Research FundThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The hybrid PLGA‐based nanoparticles as a smart nanoplatform for imaging‐guided and near‐Infrared light‐triggered combination cancer therapy
Authors: Shen Xue , Xie Xiaoxue , Yang Hong , Wu Chunhui , Liu Yiyao 
Published: June 2021

Cancer theranostics, the combination of imaging and therapeutic agents in a smart nanocarriers, has been a promising approach towards cancer [1]. And the chemotherapy combining with photothermal therapy to treat tumors can efficiently improve therapeutic efficacies [2].Herein, we report a theranostic nanoplatform based on PLGA nanoparticles, which are constructed by simultaneous encapsulating indocyanine green (ICG), a widely used near‐infrared (NIR) dye in photothermal therapy, and doxorubicin (Dox), a chemotherapy drug, into biodegradable poly(D, L‐lactic‐co‐glycolic acid) (PLGA) polymeric nanoparticles and decorated with bovine serum albumin (BSA) using the double emulsion solvent evaporation method (W/O/W). The as fabricated IDP NPs with the mean diameter of 220 nm exhibited good dispersity, excellent size stability, especially enhanced photostability (fluorescence stability) compared with free ICG, and is able to efficiently load chemotherapeutic drug doxorubicin (Dox) into the PLGA core. When the weight ratio of added ICG to Dox was 0.075 (μg ICG/μg Dox), the encapsulation efficiency of the Dox and ICG can be reached approximately 42.0% and 55.3%, respectively. It was confirmed that IDP NPs showed preferable photothermal effect, favorable photoacoustic (PA) imaging properties and faster release of Dox when exposed to near‐infrared (NIR) laser irradiation or acidic conditions. Moreover, the NIR light‐induced photothermal heat can enhance the permeability of the lysosomal membrane resulting in efficient Dox translocation from lysosomes to cytoplasm indicating by intracellular distribution assay. In comparison with the chemo‐ or photothermal treatment alone, the combined treatment of IDP NPs with laser irradiation achieving a marked synergistic therapeutic effect. The tumor cell viability was ~5% when treated with IDP NPs ([ICG] =15 μg/mL, [Dox] =3.9 μg/mL) under NIR laser irradiation (808 nm, 1.5 W/cm2). Our work validates that the developed co‐loaded BSA‐decorated IDP NPs could serve as a promising platform for PA imaging‐guided combined chemo‐ and photothermal therapy with reducing side effects in cancer theranostics.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating interactions between zebrafish innate immune system and cancer progression
Authors: Lou Meng , Powell Davalyn , Huttenlocher Anna 
Published: June 2021

In recent years, the innate immune system has been increasingly implicated in cancer prognosis. Neutrophils have been shown to migrate to the tumor microenvironment, release pro‐invasion factors, and even enhance angiogenesis. Here, we provide a glioblastoma tumor‐initiation model by transforming astrocytes with a constitutively activated form of kRasV12 in hopes of elucidating the contribution of neutrophil presence to tumorigenesis and progression. Constitutive expression of this oncogene has been demonstrated to induce malignant transformation in several models including the larval zebrafish. Additionally, the role of tumor suppressor protein p53 in relevance to this model remains largely unconfirmed. By expressing the oncogene in different p53 backgrounds, we demonstrate an increase in abnormal growth and mass formation in the absence of p53. Further experimental design perturbing neutrophil recruitment has the potential to identify novel interactions with the innate immune system and aggression of glioblastomas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.