bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tributyltin Effects on Akt/Protein Kinase B and Ribosomal S6 Protein Phosphorylation
Authors: Hamza Nafisa , Boules Mariam , Whalen Margaret 
Published: June 2021

The ERK1/2 and p38 mitogen activated protein kinase (MAPK) pathway have been shown to be activated by exposure to the environmental contaminant tributyltin (TBT). TBT is found to contaminate a number of food products that humans consume resulting in measureable levels in human blood (ranging as high as 261 nM) as well as other tissues. Recent studies have shown that TBT at certain concentrations increases the production of the pro‐inflammatory cytokine interleukin 1 beta (IL‐1β) from human lymphocytes without increasing the mRNA for IL‐1β. TBT‐induced production of IL‐1β appears to require the ERK1/2 and p38 pathways. However, it is possible that TBT may be activating other pathways such as Akt/protein kinase B (PKB) which could also contribute to its ability to increase IL‐1β production. Additionally, downstream substrates of both ERK1/2 and Akt/PKB can lead to the activation of protein synthesis in part by stimulating phosphorylation of the ribosomal S6 protein. Thus, the current study examines whether TBT is able to activate Akt/PKB and S6 phosphorylation as possible mechanisms for the TBT‐induced increases in IL‐1β production. Human lymphocytes were exposed to TBT for 10 minutes. Following the exposure the cells were lysed. The lysates were analyzed using western blot. Antibodies specific to the activated forms of Akt/PKB and S6 were used. Initial results indicate that exposure to TBT increases the activation of Akt/PKB and S6. Thus, it is possible that TBT is stimulating IL‐1β production by activating both MAPK and Akt/PKB signaling pathways leading to increased translation of IL‐1β.Support or Funding InformationSupported by NIH grant U54CA163066 and 2T34GM007663This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LRP1 requires the I3 domain of hDlg for interaction with the motor protein KIF13B
Authors: Mills Joslyn E. , Hanada Toshihiko , Liscum Laura , Chishti Athar 
Published: June 2021

KIF13B, a kinesin‐3 family motor, was identified by virtue of its biochemical interaction with human homologue of Drosophila discs large tumor suppressor (hDlg). Unlike its orthologue KIF13A, KIF13B contains a C‐terminal CAP‐Gly domain. Recently, the generation of a full‐length KIF13B null mouse model revealed a functional role in the endocytosis of LDL receptor related protein (LRP1) resulting in elevated serum cholesterol and plasma factor VIII activity. Here, we have developed two mouse models, a full‐length KIF13A null mouse and a mouse with a truncated KIF13B protein lacking its C‐terminal CAP‐Gly domain (KIF13ΔCG). To define the specific role of the CAP‐Gly domain of KIF13B in LRP1 endocytosis, we show that KIF13BΔCG mice exhibit higher levels of serum cholesterol consistent with the reduced uptake of [3H]‐LDL in KIF13BΔCG mouse embryonic fibroblasts. Alternatively, the plasma levels of factor VIII are not significantly different from wild type mice suggesting that the CAP‐Gly domain of KIF13B plays a role in lipoprotein endocytosis but not necessarily other functions of LRP1‐mediated endocytosis. We find that the truncation of KIF13B causes subcellular mislocalization of the protein and also leads to the mislocalization of LRP1, which likely contributes to the observed phenotype. To delineate the interactions of the LRP1 complex at the plasma membrane, we discovered that the cytoplasmic tail of LRP1 interacts with the alternatively spliced I3 domain of hDlg, which in turn interacts with KIF13B. Together, the LRP1‐hDlg‐KIF13B complex emerges as a novel regulator of LRP1‐mediated interactions with implications in metabolism, cell polarity, and development.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Dog, Cat, Guinea Pig, and Human IAPP toxicity in HeLa cells
Authors: Ledesma Litza Itzel 
Published: June 2021

Type 2 diabetes is one of the leading causes of death in the U.S. Type 2 Diabetes is the most generic form of diabetes, where your body suffers from insulin resistance. Islet Amyloid Polypeptide (IAPP) is a regulatory peptide which inhibits glucagon and insulin secretion. The aggregation of IAPP is present in pancreatic islet amyloid deposits seen especially in Type 2 diabetes in humans and other mammalian species. Our objective was to see how the changes in IAPP sequence correlates to the toxicity of IAPP on mammalian cells. Here, we focus on dog, cat, and guinea pig IAPP. Molecular techniques such as MTT assay and LDH Cytotoxicity assay were used to test the effect animal IAPP has on HeLa cells. As well as to see the effect mixing human and animal IAPP has on the HeLa cells. MTT assay results showed that the animal IAPP volumes had no significant effect on mammalian cell viability while the human IAPP was consistently toxic to the cells. Cell viability percentages stayed relatively constant despite increasing IAPP volume. In comparison, human IAPP showed a decreasing cell viability trend with increasing IAPP volume. However, when dog IAPP and human IAPP were mixed, cell viability was increased. The same effect occurred with the mixing of cat IAPP and human IAPP. LDH Cytotoxicity assay results showed that percent toxicity increased with increasing IAPP volumes for human, cat and guinea pig. Dog IAPP cell toxicity stayed constant as IAPP volume increased. When dog IAPP and cat IAPP were separately mixed with human IAPP, a decrease in cell toxicity is seen. Mixing guinea pig IAPP with human IAPP had no effect on cell viability or cell toxicity. Further experiments are necessary to determine a correlation between these specific animal IAPPs and cell toxicity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure and Interaction Analysis of Human R7‐RGS/Gβ5/R7BP complexes
Authors: Adikaram Poorni R. , Zhang Jianhua , Pandey Mritunjay , Kittock Claire , Simonds William F. 
Published: June 2021

The R7‐RGS/Gβ5 protein duplex functions as a GTPase‐activating protein (GAP) to facilitate the Gαi/o intrinsic GTPase activity. The R7‐RGS subfamily of RGS (Regulator of G protein Signaling) proteins comprises RGS6, RGS7, RGS9 and RGS11. The R7 family‐binding protein (R7BP) anchors the R7‐RGS/Gβ5 duplex to the cell membrane to enhance its GAP activity. Recently it was shown that knock‐out of R7BP in mice led to decreased acute and chronic itch sensation, indicating that R7BP has a central role in itch response. We are developing interfering peptides to block the R7BP and R7‐RGS/Gβ5 interactions as potential inhibitors of chronic itch. We demonstrate here the use of cross‐linking mass spectrometry (XL‐MS) to obtain structural clues about human R7‐RGS/Gβ5/R7BP triplexes. To this end, we first transfected plasmids encoding human RGS7, Gβ5 and R7BP into Expi293 cells and purified the protein triplex to 95% purity. The triplex was treated with the cleavable cross‐linking reagent DSSO (disuccinimidyl sulfoxide) at different protein:DSSO molar ratios and analyzed by mass spectrometry. The experiment was repeated using the human RGS9/Gβ5/R7BP triplex. Both experiments yielded peptides involved in the RGS7/9‐R7BP interaction, with one R7BP peptide common to both. We believe this peptide region to be an essential interaction surface on R7BP and an ideal target for interfering peptide design to block the R7‐RGS/R7BP interaction. Taken together, we demonstrate that XL‐MS is an effective way to shed light on structural features of the R7‐RGS/Gβ5/R7BP triplex to facilitate the development of novel anti‐itch drugs.Support or Funding InformationNational Institute of Diabetes and Digestive and Kidney Diseases Intramural Funding, NIHThis 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 Kinetics, Mechanism, and Reaction Pathway of a Biodesulfurizing Enzyme from Rhodococcus erythropolis, Dibenzothiophene Monooxygenase
Authors: Jirde Samatar Ali , Mendez Gilberto , Gonzalez‐Osorio Liliana , Johnson Kellee Joanne Eberle , Palfey Bruce , Vey Jessica 
Published: June 2021

Fossil fuel combustion is a major source of sulfur dioxide, dangerous to respiratory health and the environment. Sulfur removal from fossil fuels eases these effects, however, the current method, hydrodesulfurization, requires costly high temperatures and pressures. The bacterial species Rhodococcus erythropolis uses a four‐step enzymatic process, known as biodesulfurization, to remove sulfur in fuel compounds. The first enzyme in the pathway, dibenzothiophene monooxygenase (DszC), uses reduced flavin and molecular oxygen to generate a flavin intermediate that oxygenates its substrate, dibenzothiophene, to dibenzothiophene sulfoxide, then dibenzothiophene sulfone. Our objectives are to understand DszC's enzymatic mechanism and steps of the catalytic pathway to engineer DszC to accept various organosulfur compounds and increase catalytic efficiency. Towards our goals, we have purified active DszC; determined steady‐state kinetic parameters of DszC as a function of dibenzothiophene, dibenzothiophene sulfoxide, and flavin substrates; performed steady‐state experiments to describe DszC's enzymatic mechanism; and used transient‐state kinetics to ascertain the effects of dibenzothiophene and age times on flavin binding. Using affinity chromatography, we purified DszC at 0.5 gram/liter of culture in its native tetrameric state. Reverse‐phase HPLC experiments showed DszC had faster product turnover using dibenzothiophene sulfoxide as substrate versus dibenzothiophene (11.8 × 10−3 s−1 vs. 4.84 × 10−3 s−1). HPLC assays also demonstrated that having equimolar flavin and DszC resulted in a faster turnover rate than using excess flavin (4.84 × 10−3 s−1 vs. 1.49 × 10−3 s−1). Varying flavin and dibenzothiophene concentrations under steady‐state conditions indicated DszC likely forms a ternary complex with the flavin intermediate and dibenzothiophene to facilitate oxygenation. Using stopped‐flow spectrophotometry, we observed fast flavin binding (<1 second) and that having equimolar dibenzothiophene and DszC increased intermediate formation rates. Dibenzothiophene pre‐incubation with DszC, however, decreased intermediate formation rates, suggesting reduced flavin is the initial substrate to bind to DszC. These results provided us kinetic and mechanistic insights towards understanding the full catalytic pathway of DszC to support our goals of engineering a robust and efficient DszC enzyme for industrial applications.Support or Funding InformationNational Institute of Health (SCORE 5SC2AI109500)Research Corporation CCSA 22672MARC/RISE Program (NIH Grant Number 2R25GM063787‐10)CSUPERB New Investigator GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphoproteomic Analysis Identifies Dynamic Regulation of Caveolin‐1 Phosphorylation and Caveolae Formation by mTORC2 in Bladder Cancer Cells
Authors: Hau Andrew , Gupta Sounak , Leivo Mariah , Zhou Weidong , Hodge Alex , Wulfkuhle Julie , Conkright Brian , Bhuvaneshwar Krithika , Rao Shruti , Madhavan Subha , Nakashima Kazufumi , Petricoin Emanuel 
Published: June 2021

The mammalian target of rapamycin (mTOR) and associated PI3K/AKT/mTOR signaling pathway is commonly upregulated in cancer, including bladder. We previously described that mTOR complex 2 (mTORC2) is a critical regulator of bladder cancer cell migration and invasion, but the precise mechanisms by which mTORC2 regulates these processes are poorly defined. Using a dual mass‐spectrometry (MS) and reverse‐phase protein array (RPPA) proteomics screening approach to reveal novel downstream phosphoprotein targets of mTORC2 in J82 and T24 bladder cancer cells, we identified 26 and 22 significantly altered biological functions putatively upregulated or down‐regulated, respectively, by mTORC2. Represented among these functions are phosphoproteins involved in regulating cell morphology, cellular assembly and organization, cell and focal adhesion, cytoskeletal rearrangement, cellular motility and others not previously linked to mTORC2 activity. Validation of a subset of these proteins revealed the ability of mTORC2 to regulate caveolin‐1 (CAV‐1) localization and dynamic phosphorylation of CAV‐1 on Y14. Regulation of CAV‐1 activity also altered the abundance of caveolae, which are specialized lipid raft invaginations of the plasma membrane that play a critical role in cell signaling pathways associated with cell and focal adhesion, and cell motility. Our results have demonstrated the utility of proteomics‐based analyses for novel elucidation of protein targets of mTORC2.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A mechanism for signal‐dependent IKKβ activation driven by molecular interactions with poly‐ubiquitin‐bound NEMO
Authors: Cohen Samantha , Shumate Kyle , Huxford Tom , Ghosh Gourisankar 
Published: June 2021

The IκB kinase (IKK) complex is an enzyme that regulates the activation of the transcription factor NF‐κB in response to a diverse set of extracellular signals to control expression of many genes including those that mediate the cell survival program involving immune and inflammatory responses. IKK is composed of two catalytic subunits: IKK2/β and IKK1/α in addition to a non‐catalytic subunit known as the NF‐κB essential modulator (NEMO)/IKKγ. In this study, we focus on IKKβ which is the subunit primarily responsible for signal‐dependent activity in pro‐inflammatory signaling. Like many protein kinases, IKKβ activation is achieved through phosphorylation of two serine residues (S177 and S181 in IKKβ) located within its activation loop. However, the molecular details of how the activation loop serine residues undergo signal‐induced phosphorylation is still unknown. The x‐ray crystal structure and mutational analyses from our laboratory identified specific crystallographic interfaces (V‐shaped and KD‐KD interfaces) that are critical for IKKβ activation. Disruption of these interfaces reduced the ability of IKKβ to be activated in cells, suggesting the significance of protein‐protein contacts in activation. It is known that an N‐terminal segment of NEMO comprising the IKKβ binding domain (IKKBD) is responsible for a stable interaction with the C‐terminal NEMO binding domain (NBD) of IKKβ. Here, we propose a mechanistic model of IKKβ activation which invokes a transient second site interaction between the NEMO Ubiquitin binding in ABIN and NEMO (UBAN) domain and the IKKβ V‐shaped interface via a ubiquitin (Ub)‐chain. Engagement of poly‐Ub chain‐bound NEMO to IKKβ at this second site leads to conformational changes of the activation loop in an allosteric manner, resulting in the phosphorylation of S177 and S181. Cell transfection studies using V‐shaped interface mutants indicates that the second site of interaction critical for IKKβ activation coincides with the V‐shaped interface in a NEMO‐dependent manner. In vitro binding assays show that the NEMO UBAN (which lacks the IKKBD) binds to IKKβ (devoid of the NBD) in the presence of poly‐Ub. In vitro activation assays reveal that the activation loop is phosphorylated in an ATP, NEMO UBAN, and poly‐Ub‐dependent manner. We conclude that the molecular interactions between the V‐shaped interface of IKKβ and the poly‐Ub‐bound NEMO UBAN is responsible for signal‐dependent IKKβ activation loop phosphorylation. Our biochemical and cell‐based results give a detailed molecular understanding behind IKKβ activation in pro‐inflammatory signaling.Support or Funding InformationThis project was funded by an NIH R01 grant to Gourisankar Ghosh and supported by the California Metabolic Research Foundation at San Diego State University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing Professionalism in Research through Journals Clubs
Authors: Mendez Gilberto , Prokopczuk Federico 
Published: June 2021

Professional development in science begins with knowing how to communicate science effectively. Journal clubs are a great tool in supporting student's exposure to science and the way it is communicated to others. Breaking down articles helps improve a person's ability to critically appraise research while at the same time unveiling the vast world of scientific research. The California State University, Northridge (CSUN) ASBMB Student Chapter was established in 2017 to promote professionalism in biochemistry and molecular biology research. This chapter is based on specialized formal education activities, such as student‐led journal clubs to give participants the opportunity to develop essential skills and knowledge needed for a successful life as a future scientist. The structure for our journal clubs is very interactive: all participants must come to the meeting having read the paper, and a discussion leader is assigned beforehand to facilitate conversation. The leader primes the students with introductory knowledge on the subject and all students are assigned tasks while the leader asks the participants to answer specific questions on the concepts, figures, and discussion. Participants are free to ask questions and aid in the discussion throughout the meeting. Distinguished professors and researchers are also invited to give insight and an experienced perspective. The primary focus of the discussion is not to reiterate data, but instead understand experimental strategy and rationale. Journal clubs are hosted eight times a semester, and all members must be actively participating to maintain membership in the chapter. This method serves to ensure that all members improve their professional knowledge, competence, skill, and effectiveness in research. The club saw much success with this approach during our first semester, with nearly all 19 members saying that they saw much progress in their critical thinking and ability to communicate science. This experience is a gateway for many, if not all of the students who have never experienced this level of scientific engagement and discourse. Our mission is to help break the barriers of fear and self‐doubt that comes with delving into science, one journal article at a time.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of Gα13 exercise‐mimetically reprograms skeletal muscle through Rock2
Authors: Kim Sang Geon , Koo Ja Hyun 
Published: June 2021

Skeletal muscle is a key organ in energy homeostasis owing to its high requirement for nutrients. Heterotrimeric G proteins converge signals from cell‐surface receptors, potentiating or blunting responses against environmental changes. Here, we report that muscle‐specific deletion of Gα13 promotes reprogramming of myofibers to oxidative type, with resultant increases in mitochondrial biogenesis and cellular respiration. Mechanistically, Gα13 and its downstream effector RhoA suppressed nuclear factor of activated T‐cells 1 (NFATc1), a chief regulator of myofiber conversion, through Rho‐associated kinase 2 (Rock2)‐mediated phosphorylation at Ser243. Ser243 phosphorylation of NFATc1 was reduced after exercise, but was higher in obese animals or diabetic patients. Consequently, Gα13 deletion in muscles enhanced whole‐body energy metabolism and increased insulin sensitivity, thus affording protection from diet‐induced obesity and hepatic steatosis. Our results define Gα13 as a switch regulator of myofiber reprogramming, implying modulations of Gα13 and its downstream effectors in skeletal muscle as a potential therapeutic approach to metabolic diseases.Support or Funding InformationThis work was supported by grants from the National Research Foundation of Korea (NRF), funded by the Korean government (MSIP) (2017K1A1A2004511).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepatocyte Specific Shp1 Deletion and Low Dose Rosiglitazone Treatment Act in Concert to Improve Liver Glucose Homeostasis in Diet‐induced Obese Mice
Authors: Leandro Joao Gabriel Bernardo , Forest Marie‐Pier , Lavallée‐Bourget Marie‐Hélène , Gomez Lilian , Moreira Rafael , Houde Vanessa , Schwab Michael , Bellmann Kerstin , Deshaies Yves , Sola‐Penna Mauro , Marette André 
Published: June 2021

The protein‐tyrosine phosphatase Shp1 plays a key role in high‐fat diet (HFD)‐induced insulin resistance by negatively regulating insulin action on glucose homeostasis. Hepatocyte‐specific Shp1 knockout mice (Ptpn6H‐KO) on HFD show an improvement of hepatic insulin resistance and glucose homeostasis but also develop increased hepatic steatosis compared to their wild‐type littermates (Ptpn6fl/fl). Nevertheless, livers of Ptpn6H‐KO mice on HFD have reduced hepatocellular damage and a reduced inflammatory profile alongside with increased peroxisome proliferator‐activated receptor gamma (PPARγ) activity, suggesting a new role for Shp1 in the regulation of PPARγ and the control of hepatic lipid metabolism. Therefore, the aim of the present study was to determine whether activation of PPARγ activity using rosiglitazone could further ameliorate hepatic glucose and lipid homeostasis as well as the inflammatory profile in Ptpn6H‐KO mice. Male Ptpn6H‐KO and Ptpn6fl/fl mice were fed a HFD for 18 weeks. During the last 4 weeks the animals were treated with a very low dose of rosiglitazone (0.3 mg/kg/day). Low‐dose rosiglitazone treatment did not affect total body or adipose tissue weight, plasma levels of adiponectin or expression of PPARγ target genes in adipose tissue whereas PPARγ target genes expression was highly increased in liver. However, liver weight and liver triglycerides were increased in both rosiglitazone treated groups while plasma triglycerides were reduced by rosiglitazone. Furthermore, treatment with rosiglitazone led to an improvement in whole‐body glucose homeostasis and hepatic Akt activation both of which were even more ameliorated in HFD‐fed Ptpn6H‐KO compared to Ptpn6fl/fl mice. These results show that a low‐dose rosiglitazone treatment selectively target liver metabolism without detectable effects on adipose tissue, and our data further suggest that hepatic invalidation of Shp1 acts in concert with rosiglitazone to improve glucose metabolism in liver of HFD‐fed obese mice.Support or Funding InformationCIHRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.