bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reconstitution of P‐glycoprotein in Nanodiscs using the MSP1D1 Scaffold Protein for Biochemical Inhibitor Screens
Authors: Ballou‐Crawford Jett J. , Chen Gang , Oliveira Maisa C. , Wise John G. , Vogel Pia D. 
Published: June 2021

Many cancers become multidrug resistant due to overexpression of the transmembrane efflux pump P‐glycoprotein (P‐gp). Compounds inhibiting P‐gp function may therefore have the potential to improve the effectiveness of chemotherapeutic drugs. Using in silico methods, we have been able to successfully identify a number of putative P‐gp inhibitor compounds. In cell culture assays, many of these compounds were shown to re‐sensitize cancers to chemotherapeutics. However, when tested with purified P‐gp, some of these compounds showed little effect on the catalytic activity of the isolated protein. The relatively high concentrations of detergents needed to solubilize P‐gp likely reduced the potency of the inhibitor compounds. To overcome this issue, purified P‐gp was inserted into nanostructures (nanodiscs) which much more closely resemble natural cell membrane. We and others have observed that this stabilizes P‐gp and stimulates its catalytic activity. However, previous assays of inhibitor compounds with medium size MSP1E3D1 nanodiscs still did not show clear effects on catalytic activity of the protein despite the fact that the inhibitors were effective in cell culture. We hypothesize that the previously used MSP1E3D1 nanodiscs still may have been too large, leaving hydrophobic space into which the inhibitors could partition, again reducing the effective concentration of the inhibitors. To improve on this protocol, we here show our efforts to use a shorter membrane scaffold protein, known as MSP1D1, for the assembly of smaller nanodiscs. After optimization of conditions for assembly and insertion, and tests for biological activity, we will assay our potential inhibitor hit compounds for their ability to affect catalytic ATP hydrolysis activities of P‐gp.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars Program, SMU Undergraduate Research Assistantships, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Retinoblastoma‐derived peptides directly inhibit the CMG helicase and impair proliferation of cancer cells
Authors: Larratta Auston J. , Borysov Sergiy 
Published: June 2021

The CMG helicase is the main replicative helicase. Circumstantial evidence suggests that interference with the function of this helicase is detrimental for cancer cells, while tolerable for normal healthy cells, pointing to the CMG helicase as an ideal novel target for cancer treatment. Previous studies identified Retinoblastoma (Rb) protein as a potent native inhibitor of the helicase activity. For the purpose of drug discovery, here we explore Rb‐derived peptides for their abilities to inhibit the CMG helicase and proliferation of cancer cells. For the first time, we showed that Rb‐derived peptides directly inhibited activity of human CMG helicase, as assessed in an in vitro helicase assay. Importantly, Rb‐derived peptides caused a substantial decrease in the proliferation of cancer cells in vitro. Our results further support the idea of targeting CMG helicase for cancer treatment and developing novel anti‐cancer therapeutic agents based on Rb‐derived peptides.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Purified Plant Extracts and Homologous Commercial Derivatives on Entamoeba histolytica growth
Authors: Hunt Sara , Santos Yoly , Seeram Navindra , Rowley David , Ma Hang , Espinsosa Avelina 
Published: June 2021

Entamoeba histolytica is a parasitic protist which causes amoebiasis in humans. Amoebiasis causes about 100,000 deaths every year worldwide. Currently, the antibiotic, metronidazole is the only effective treatment for amoebiasis. Metronidazole is effective; however, it has a high toxicity and causes damage to the nervous system. Finding an alternative treatment is necessary to manage the disease better and reduce the risks associated with the current medication. In previous studies, purified plant extracts and homologous commercial derivatives have shown significant antimicrobial character. Previously, we identified pomegranate, rhubarb (rhein), and maple syrup (maplifa) extracts as potential inhibitors of E. histolytica trophozoite growth. The effectiveness of the extracts was assessed by growth inhibition assays monitored at 12, 24, and 48 hours using 60 μM or 120 μM concentrations of pure compounds. Rhein and maplifa, showed promising inhibitory activity on E. histolytica trophozoite growth. Testing of compounds of analogous structures is currently in progress. Future studies will be completed to determine the biochemical mechanism of action of each of these inhibitors and test their toxicity to mammalian tissue cultures in comparison to the current drug of choice, metronidazole.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards the Identification of the Minimal Pharmacophore of KDT‐11
Authors: McCartney Julia , Dickson Paige , Kodadek Thomas 
Published: June 2021

The 26S proteasome is a key player in a number of cell processes that help to remove misfolded and downregulate targeted proteins. Rpn13, a subunit of the 19S regulatory particle of the proteasome, is a ubiquitin receptor and UCH37 recruiter and activator. Rpn13 can be knocked out of yeast and mouse models without the development of a new phenotype, yet in many cancer cells it is over expressed. In 2015, the peptoid KDT‐11 was shown to be highly selective towards Rpn13 and toxic to multiple myeloma cells. However, because of this compound's size and hydrophobicity, it is a poor candidate for further development as a drug. Preliminary lab data suggests that N‐methyl‐N‐(3‐fluorobenzylamine) in the terminal position of the 6‐mer peptoid is critical for selective toxicity. However, the necessity of the amines in all other positions is not known. To make the molecule more biologically viable, I synthesized ten derivatives using solid‐phase bead synthesis. Florescence polarization and cell viability assays were conducted to assess the potential improvement or detriment offered by each variation, using KDT‐11 and Bortezomib, a 20S core particle inhibitor, as control factors. I found that shortening the peptoid does not inherently result in a loss of selectivity or cytotoxicity, and in some cases, appears to lead to an increase. However, truncating past a 4‐mer length resulted in general cytotoxicity to control and cancerous cells. These data indicate that while a certain length must be maintained, only the terminal N‐methyl‐N‐(3‐fluorobenzylamine) is position dependent. This development is highly advantageous as it targets a different subunit of the proteasome than currently available drugs, and could potentially be used in parallel.Support or Funding InformationWe would like to thank the Scripps Summer Undergratuate Research Fellowship for funding this workThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exon Sequencing for the Presence of Late Onset Tay‐Sachs
Authors: Slostad Joshua Nathan , Goldberg Jodi 
Published: June 2021

Tay‐Sachs is a disease that causes rapid developmental stagnation and death in infants. It primarily affects people of French Canadian, Cajun, and Ashkenazi Jewish descent. In recent years an alternate form of the disease, called late onset Tay‐Sachs (LOTS), has been described. LOTS is caused by the inheritance of one null allele and a pseudodeficiency allele of the hexosaminidase A (HEXA) gene. The mutation rate of the pseudodeficiency is not well known. The aim of this experiment was to examine the DNA of the descendants of an Ashkenazi Jewish individual who was believed to have undiagnosed LOTS. The purpose of this case study is to attempt to confirm this suspected diagnosis by identifying potential mutations in the family members. DNA of the HEXA gene from three family members of the suspected LOTS patient (wife and 2 daughters) and one control set (unrelated female) was sequenced. The sequences were aligned and compared both by hand and using the NCBI Blast database and the Benchling tool. No known mutations or confirmed novel mutations have been found in all fourteen exons sequenced. Currently re‐sequencing of some regions that showed potential single nucleotide polymorphisms (SNPs) as well as sequencing the promoter region of the gene is underway. If a mutation is confirmed this research could help pave the way for further genetic testing in the broader population in order to fully grasp the prevalence of LOTS pseudodeficiency alleles within the Ashkenazi population.Support or Funding InformationHamline Undergraduate Collaborative ResearchRidgeway Forum FundThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thermo‐responsive Estrogen Receptor‐fusions for Spatiotemporal Control of Signaling and Gene Regulation
Authors: Truong Anh T. , Li Zhe , Okamoto Curtis T. , MacKay J. Andrew 
Published: June 2021

Many diseases are caused by aberrant cell signaling, leading to a dysregulation of proteins responsible for cell maintenance and repair. Understanding these events require a precise manipulation of the cells' machinery, but many technologies used to study signaling and transcription lack the temporal control to rapidly modulate these biologic processes. This limitation could be addressed by the introduction of elastin‐like polypeptides (ELPs) which are synthetic, thermally responsive biopolymers that self‐assemble into microdomains of ELP‐fusions within cells. Our research group recently discovered that these fusion proteins can modulate precise processes involved in cell trafficking. They are now being investigated to modulate estrogen receptor (ER) signaling and gene regulation. ER has been implicated in a number of diseases such as breast cancer and Sjögren's Syndrome and although this nuclear hormone receptor has been well studied, the lack of drug therapy and resistance, requires attention to develop a deeper understanding of the underlying mechanisms. Thus, ELP‐ERɑ fusions have been generated and successfully expressed in CHO, HeLa and 293T cell lines. They coalesce in the nucleus at physiological temperatures and rapidly solubilize at room temperature. These preliminary data indicate that these fusions retain ELPs self‐assembling properties and may control ER signaling and gene regulation. The ability to rapidly and reversibly, with spatiotemporal control and precision, may allow this fusion to serve as a powerful tool to study ER‐mediated biological processes.Support or Funding InformationThis funding is supported by the National Institutes of Health (NIGMS 1R01GM114839‐02) and the American College of Clinical Pharmacy Research Institute Futures Grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Depletion of CRBN leads to proliferative cellular senescence by altering the level of cell cycle proteins
Authors: Jeon Seung‐je , Yoon Yi‐seul , Yang Seung‐joo , Park Chul‐Seung 
Published: June 2021

Cereblon (CRBN) is the direct target of the immunomodulatory drugs (IMiD), such as lenalidomide, and the treatment of IMiDs is known to inhibit the proliferative activities of cancerous cells such as multiple myeloma. The expression level of CRBN protein is varied in different cancer cells and many somatic mutations in CRBN gene are found in numerous cancer patients. The mouse embryonic fibroblast (MEF) lacking Crbn was previously shown to be resistant to various cellular stresses (K.M. Lee et al. / Biochemical and Biophysical Research Communications 458 (2015) 34–39). However, the role of CRBN in cell proliferation and its regulation is still poorly understood. We observed that the Crbn‐knockout (KO) MEFs showed accelerated cell proliferation, and the increased level of replicative and premature senescence. We thus evaluated the level of cell cycle proteins such as cyclins, cyclin‐dependent kinases (CDK), and CDK inhibitors. We found that the expression of several cell cycle proteins was specifically altered by depletion of CRBN protein. In addition, the flow cytometric analyses showed the increased level of cell arrest at G2/M phase for CRBN KO cells by. Furthermore, the lifespan of Crbn KO mice was found to decrease compared to that of WT mice. Thus, CRBN deficiency may induce proliferative senescence via modulating the level of cell cycle proteins such as cyclins and CDK inhibitors. We are currently investigating the underlying mechanism for the specific alteration of cell cycle proteins induced by CRBN depletion.Support or Funding InformationThis work was supported by GIST Research Institute(GRI) grant funded by the GIST in 2017.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Caveolin‐1 Phosphorylation is Essential for iPSC‐derived Human Neuronal Axonal Growth during Early Stage of Differentiation
Authors: wang Shanshan , Zhang Zheng , Leem Joseph , Patel Hemal , Head Brian 
Published: June 2021

Proper axonal growth and guidance is essential for functional neuronal connections. Formation of aberrant neuronal connections due to genetic or epigenetic influences can contribute to neurological and mental disorders. Identification and targeting of the molecular events that promote normal neuronal growth and differentiation may promote structural and functional neuroplasticity and improve behavior in neurodegenerative diseases such as Alzheimer's disease (AD), Down syndrome, schizophrenia, and depression. Using human neurons derived from induced pluripotent stem cells (iPSCs), we show that neuron‐specific targeted (synapsin promoted, Syn) caveolin‐1 (Cav‐1) overexpression (SynCav1) significantly augmented axonal growth (SMI 31), findings akin to effects from SynCav1 murine neurons in vitro and in vivo. In contrast, neurons infected with a Cav‐1 phosphorylation mutant (SynCav1(Y14A)) exhibited stunted axonal growth. In addition, SynCav1(Y14A) significantly impaired Rac1/Cdc42CA‐mediated axonal growth. These results suggest that 1) P‐Cav‐1 is necessary for proper axonal growth during its early differentiation stage, 2) SynCav1‐mediated axonal growth is dependent upon Cav‐1 phosphorylation, 3) Rac1/Cdc42CA‐mediated neuronal growth may in part be dependent upon P‐Cav‐1. In conclusion, phosphorylation of Cav‐1 is crucial for axonal growth during early stage of differentiation in human neurons.Support or Funding InformationNS073653 (B. P. Head); Veteran Affairs Merit Award from the Department of Veterans Affairs BX003671This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
c‐JUN N‐terminal kinase (JNKα1) binds eNOS and phosphorylates at S116, differentiating it from p38 and ERK phosphorylation
Authors: Solone Xzaviar K. , Caldara Amber L. , McMurry Jonathan L. , Chrestensen Carol 
Published: June 2021

We have previously shown that the mitogen activated protein kinases (MAPK), p38 and ERK bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via interactions with a pentabasic non‐canonical MAPK binding sequence in the autoinhibitory insertion of eNOS. The neuronal isoform, which lacks the pentabasic motif, did not bind MAPK. In the present study, eNOS binding by c‐Jun N‐terminal kinase (JNKα1) was examined using optical biosensing. Similar to p38 and ERK, JNK evinced binding to eNOS (31 nM KD) but not nNOS as measured by fit to a one‐state global model. Rate constants were determined (kon = 4125 M−1s−1 and koff = 1.3 × 10−4 s−1). In vitro kinase assays and immunoblotting identified phosphorylation by JNK at S116, contrasting with ERK, which phosphorylated S602, and p38, which phosphorylated both sites. Phosphorylation by JNK at S116 did not alter the conformational manifold, where phosphorylation at S602 by ERK and p38 does. In HMEC1 cells we observe that phosphorylation at these MAPK sites happens concurrently with activating sites (S1177), creating antagonistically phosphorylated eNOS. Further, we examine how phosphorylation of eNOS alters the cellular interactions of the MAP kinases. These results underscore the importance of MAPK interactions with eNOS showing that each MAPK creates a unique phosphorylation pattern. These findings strengthen the emerging paradigm of eNOS as a junction of multiple signaling pathways.Support or Funding InformationNIH 1 R15 GM110634‐01A1 | R25GM111565This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of Gap Junctional Coupling by Secreted Factor(s) from Mesothelial Cells is Required for the Invasiveness of Endometriosis
Authors: Nicholson Bruce J. , Chavez Jeffery B. , Dorsey Emma K. , Olivas Taryn , Wang Chiou‐Miin , Lucio Nicholas , Gillette Laura , Burney Richard O. , Kirma Nameer 
Published: June 2021

Intercellular interactions, including gap junction intercellular coupling (GJIC), have been implicated in many invasive processes (e.g. extravasation, metastasis and oocyte implantation). Yet the role of gap junctions in endometriosis, a major invasive medical problem that affects 6–10% of women worldwide, has not been investigated. Comparisons of the expression profile of endometrial cells (ECs) from endometriosis patients and normal subjects revealed a consistent reduction in the expression levels of connexins (the protein components of gap junctions), including the most abundant isoform, Cx43. This was most notable in stromal cells as the disease progressed, but was also evident in epithelial cells. GJIC of ECs from endometriosis patients was also reduced compared to normal subjects, again most notably in stromal cells. In mimicking the endometriosis process, heterotypic interactions with mesothelial cells induced an increase in GJIC, with stromal cells from endometriosis patients showing the largest response. This enhanced coupling correlated with enhanced trafficking of Cx43 to the cell surface, and enhanced invasiveness through a mesothelial monolayer. Blocking GJIC between endometrial and mesothelial cells using either siRNA or a Cx43 specific peptide blocker dramatically reduced this enhanced invasiveness of endometriosis derived ECs, whether epithelial or stromal in origin. Finally, GJIC induction by mesothelial cells was shown to be mediated by a secreted factor, which is heat resistant and approximately 5–10K is size. This presents a unique opportunity for developing novel therapy for treating endometriosis that would prevent the formation of lesions that cause the pain and incidence of infertility associated with this widespread disease. Currently the only therapies are based on hormonal treatments that only further compromise fertility problems associated with endometriosis (representing 35% of the cases on infertility in the nation).Support or Funding InformationWomen's Health Initiative at UTHSA School of Medicine and CPRIT grants RP150600 and RP160844, and NIH grant UL1 TR001120 for CoresThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ANXA5 Suppresses COX‐2 Expression by Downregulating the PKC‐ζ–NF‐κB Signaling Pathway in Prostate Cancer Cells
Authors: Baek Hyoung Seok , Kwon Yeo‐Jung , Ye Dong‐Jin , Chun Young‐Jin 
Published: June 2021

Annexin A5 (ANXA5) is a member of the annexin family of proteins. Previous studies showed that ANXA5 is involved in anti‐inflammation and apoptosis. However, detailed mechanisms of the role of ANXA5 in cancer cells are less well explored. In this study, we aimed to identify the cyclooxygenase‐2 (COX‐2) suppressive function of ANXA5 in prostate cancer cells. TNF‐α‐induced COX‐2 expression was suppressed by ANXA5 overexpression, and suppression of COX‐2 expression by auranofin, which may induce ANXA5 expression, was recovered by ANXA5 knockdown. In addition, ANXA5 knockdown induced phosphorylation of NF‐κB p65 in prostate cancer cells, indicating that ANXA5 causes COX‐2 downregulation via suppression of p65 activation. We also found that protein kinase C (PKC)‐ζ protein levels were upregulated by inhibition of ANXA5, although the mRNA levels were unaffected. We confirmed that upregulated COX‐2 expression caused by the inhibition of ANXA5 was suppressed by PKC‐ζ siRNA. In summary, our study demonstrates that downregulation of PKC‐ζ‐NF‐κB signaling by ANXA5 may suppress COX‐2 expression in prostate cancer cells.Support or Funding InformationNRF‐2015R1A5A1008958This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CYP1B1 induces tumorigenic metastasis through promotion of uPAR and subsequently activation of uPAR signaling via regulation of MDM2‐uPAR system
Authors: Kwon Yeo‐Jung , Ye Dong‐Jin , Baek Hyoung‐Seok , Chun Young‐Jin 
Published: June 2021

Human CYP1B1 is known as a major metabolizer for estrogen and shows tumor‐specific hyper‐expression. To explore the role of CYP1B1 on metastasis of human cancer cells, we studied the effects of CYP1B1 inhibition and activation in MCF‐10A, MCF‐7, MDA‐MB‐231, and HeLa cells. CYP1B1 significantly induced expression of urokinase‐type plasminogen activator receptor (uPAR) as well as uPA. Treatment with DMBA, a well‐known CYP1B1 inducer, enhanced uPAR expression while CYP1B1 siRNA and TMS (tetramethoxystilbene), a specific CYP1B1 inhibitor, suppressed uPAR. We also found that CYP1B1 activated uPAR signaling through regulation of related factors including integrin β1 and α5. Interestingly, uPAR overexpression only caused a significant increase of integrin β1 and α5 in protein levels, indicating protein degradation may play an important role in regulating integrin protein level. Surprisingly, CYP1B1 down‐regulated p53 expression through MDM2 activation and nutlin‐3a, an inhibitor for MDM2, blocked the promoting effects of DMBA on uPAR, which explains that CYP1B1 induced uPAR expression through regulation of MDM2‐p53 system. Taken together, our data suggest that CYP1B1 promotes cancer cell metastasis via activating uPAR pathway which is one of the targets of p53 signaling.Support or Funding InformationThis research was supported by a National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (NRF‐2015R1A5A1008958).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.