bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of High‐throughput Assays for Testing of Potential Inhibitors of the Oncogenic G12C Mutant of KRas
Authors: Vertessy Beata G , Nyiri Kinga , Steger Anett , Koppany Gergely
Published: June 2021

Objective and Background

We aim at creating and optimizing in vitro assay methods to follow binding and potential covalent linkage of small drug‐like compounds to the G12C mutant of KRas. Oncogenic mutations of Ras proteins are found in numerous cancers. The glycine 12‐position in KRas is known to harbor several oncogenic mutations, such as cysteine, aspartate and valine. All of these mutations efficiently prevent GAP binding to mutant KRas thereby resulting in a constitutively active GTP‐bound KRas conformation that drives oncogenic processes. High‐throughput assays can facilitate identification of novel drugs that specifically target the GDP‐bound inactive KRas mutant and lock it in this inactive conformation, such as eg the recently reported ARS‐853 (1).

Methods

KRas wild type and G12C was expressed and purified. Assays for thermal stability of proteins and their complexes were run on 96‐well plates using Thermofluor in a real‐time PCR instrument. Fluorescent‐based assays for thiol reactivity and (mant) GTP/GDP exchange were run on 384‐well or 96‐well plates, respectively, using a plate reader equipped with kinetics module.

Results

Thermofluor assays showed that a distinct and well‐reproducible upward shift in the thermal melting point of KRas G12C upon binding to ARS‐853. The thiol‐reactivity assay was optimized with regards to protein concentration, since we found only about 25% of the expected signal was observable. The GTP/GDP exchange assay indicated that evaluation of the kinetic parameters of the exchange assay are essential to characterize functional effects of ARS‐853 that are not revealed in simple equilibrium end‐point titrations.

Conclusions

Combination of assays focusing on protein stability, thiol reactivity and nucleotide‐exchange resukt in a complex test system that can distinguish drug‐like compounds that bind to KRas with or without functional effect. This test system is proposed for testing compound libraries.

Support or Funding Information

Supported by the National Research, Development and Innovation Office, Hungary, project number NVKP‐16‐1‐2016‐0020

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Autophagy Regulates Bile Acid Metabolism via a NRF2‐FXR Signaling Axis
Authors: Khambu Bilon , Yan Shengmin , Huda Nazmul , Liu Gang , Hong Honghai , Chen Xiaoyun , Yin Xiao‐Ming
Published: June 2021

Autophagy is a vital intracellular degradation and recycling process which is required for hepatic metabolic homeostasis. Deficiency of autophagy in the liver causes severe injury that may be causatively related to disturbed metabolism. However, it is not clear how autophagy mechanistically contributes to hepatic metabolism and whether the disturbed hepatic metabolism could be a contributing factor for hepatic injury. Here we found that hepatic deletion of Atg7 or Atg5 resulted in dysregulated bile acid (BA) metabolism, with a downregulation FXR functionality. Reduced FXR resulted in intrahepatic cholestasis due to reduced expression of Bsep, a key bile acid transporter at the apical surface of hepatocytes. Further studies indicated that an excessively high level of NRF2 could transcriptionally repress the expression of FXR. Pharmacologically or genetic activation of FXR in autophagy‐deficient liver corrected the BA metabolism and hence alleviated the cholestatic liver injury. Thus, excessive activation of Nrf2 mediated FXR repression is responsible for the dysregulation of BA metabolism and hence the cholestatic liver injury. This exciting new finding suggests that autophagy regulates hepatic metabolism beyond nutrients breakdown by controlling the NRF2‐FXR signaling components.

Support or Funding Information

Department of Pathology and Laboratory Medicine, Indiana University School of Medicine

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inflammatory cell death‐related proteins as potential biomarkers in Takotsubo syndrome
Authors: Borodzicz Sonia , Czarzasta Katarzyna , Wojciechowska Małgorzata , Puchalska Liana , Główczyńska Renata , Kołodzińska Agnieszka , Opolski Grzegorz , Cudnoch‐Jędrzejewska Agnieszka
Published: June 2021

Background

Takotsubo syndrome (TS), triggered by emotional or physical stress, is prevalent in postmenopausal women and is characterized by an acute and usually reversible left ventricular (LV) wall motion abnormalities. Histopathological analyses of TS animal models revealed presence of single cardiomyocyte cell death with features of inflammatory reaction, however the type of cell death in TS and its impact on the course of the disease has not been explained so far.

Purpose

The aim of the study was to evaluate the plasma concentration and release dynamics of proteins associated with inflammatory cell death (pyroptosis, necroptosis) and apoptosis in female rat model of TS.

Methods

9‐weeks old Sprague Dawley rats were ovariectomized and after 3 weeks received an intraperitoneal injection with a single dose of 150 mg/kg isoprenaline (TO group). The control rats were ovariectomized and received an injection of 0.9% NaCl (CO group). Echocardiography and blood collection was performed 6 (TO6h), 12 (TO12h), 24 (TO24h), 72 (TO72h) hours, and 10 (TO10d) days after ISO or NaCl administration. The plasma concentrations of cell death‐related proteins including: interleukin 1 beta (IL1b) and caspase 1 (casp1) involved in pyroptosis, caspase 3 (casp3) in apoptosis, mixed lineage kinase domain like pseudokinase (MLKL), receptor‐interacting serine/threonine‐protein kinase 1 (RIPK1) and calcium/calmodulin‐dependent protein kinase type II delta chain (CAMK2d) in necroptosis were evaluated.

Results

The echocardiographic examination in TO group revealed several types of left ventricular wall motion abnormalities, including basal, mid‐basal and apical akinesia, as well as a mixed variant. In comparison to the CO group, the median plasma concentration of IL1b was significantly higher in the TO6h (p<0.01), TO12h (p<0.01), TO24h (p<0.05) and TO72h (p<0.001) series. Moreover, when compared to the CO group, the median plasma concentration of CAMK2d was significantly higher in TO6h (p<0.001), TO12h (p<0.001), TO24h (p<0.001), TO72h (p<0.001) and TO10d (p<0.05) series. No significant differences in mean concentrations of casp1, casp3, MLKL and RIPK1 between CO and TO groups were found.

Conclusions

Significant variability of concentration and release dynamics of inflammatory cell death‐related proteins, including pyroptosis and necroptosis, indicates their potential role in pathogenesis of TS and suggests the potential use of them as biomarkers in TS.

Support or Funding Information

The research is funded by “Diamond Grant” from the Polish Ministry of Science and Higher Education (project No. DI2015 003045).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Roles of the Essential Proteins Dib1, Prp31, Prp6 and the U5 snRNA During Splicing
Authors: Embry Addie , Potts Camille , Jamison Danielle , Turner Caitlyn , Maeder Corina
Published: June 2021

Pre‐mRNA splicing is a conserved biological process essential to all eukaryotes. Splicing defects lead to a variety of disorders in humans including Retinitis Pigmentosa and Burn‐McKeown Syndrome, defining the importance of splicing in health science fields. Pre‐mRNA splicing requires complex machinery composed of five small nuclear RNAs (snRNAs) complexed with ~90 proteins that form small nuclear ribonucleoproteins. Spliceosome assembly occurs over several dynamic steps to form a pre‐catalytic B complex before splicing reactions can occur.

Splicing cannot begin until the pre‐mRNA and U5 snRNA can interact. Analysis of cryo‐EM structures (Plaschka, et al. 2017) as well as biochemical and genetic studies reveal three essential proteins in the B complex blocking a pre‐mRNA‐U5 snRNA interaction. At the center of the catalytic site sits Dib1, a 17‐kDa protein with a thioredoxin‐like (TRXL) domain. Prp6 is a large, 102‐kDa U4/U6.U5 triple‐snRNP protein whose N‐terminal region appears to interact with the hydrophobic pocket located on one side of the Dib1 TRXL domain. Prp31 is another large, 56‐kDa protein that appears to interact with a basically charged region of Dib1 opposite of the hydrophobic pocket via acidic residues on its extended loop region. Crosslinking studies (Dix, et al. 1998) revealed that the U5 snRNA loop 1 interacts with a 16‐kDa protein and cryo‐EM studies of B complexes revealed U5 loop 1 may interact with Dib1.

Prp6, Prp31, and Dib1 must all depart the spliceosome before splicing can occur. The interactions between these proteins and the U5 snRNA may serve to stabilize the spliceosome and prevent the premature splicing of pre‐mRNA. Here, we aim to identify temperature sensitive (ts) growth mutants of Prp6, Dib1, Prp31 and U5 snRNA that inhibit or abolish splicing and identify the mutants' roles in the assembly of the B complex and the U4/U6. U5 triple snRNP. Studies are performed in S. cerevisiae, an excellent model to study splicing. Our findings of key residues in Prp6, Prp31 and U5 snRNA that are important for interactions with Dib1 will be presented as well as the effects of these novel mutants on splicing and spliceosome assembly.

Support or Funding Information

NIH‐NIGMS R15GM120720 and the Welch Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Influence of PIP2 on IDE and UCP2 in RIN‐5F Cells”
Authors: Clemons Tameka Arlene
Published: June 2021

Phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) is present on the plasma membrane of cells and is the precursor to inositol 1,4,5‐trisphosphate (IP3) and diacylglycerol (DAG). Although PI(4,5)P2 has traditionally been considered the substrate for phosphatidylinositol 3‐kinase (PI3Kinase) or phospholipase C (PLC), PI(4,5)P2 has recently been shown to bind Insulin Degrading Enzyme (IDE). IDE is a cytosolic protein that has been shown to be anchored by PI(4,5)P2 until it is ready to perform its function as a protease. IDE serves as a protease for several proteins including insulin and amylin. Insulin and amylin are two proteins that are co‐secreted from pancreatic β cells after a rise in cellular glucose levels. The degradation of insulin and amylin occur by IDE after the two proteins have completed respective actions, most notably the lowering of glucose levels and the increase in satiety. A rise in glucose is known to lead to a rise in insulin and amylin levels in order to maintain metabolic homeostasis. Uncoupling Protein 2 (UCP2) is a mitochondrial membrane protein that is known for its impact on the oxidative phosphorylation process. The presence of UCP2 on pancreatic β cells has previously been shown to have an inverse relationship with insulin. Here we conducted IDE, amylin, and UCP2 ELISA assays before and after inhibition of PLC or PI3Kinase under varying concentrations of glucose in RIN5F cells, which is a rat pancreatic β cell line, in order to investigate the impact of IDE on the activity level of amylin and UCP2 and to establish a possible relationship between amylin and UCP2. In this study, we measured IDE, amylin and UCP2 levels with the PLC inhibitor, U73122, and PI3kinase inhibitor, wortmannin, in comparison to the negative controls. The initial data shows that a decrease in IDE occurs in the presence of the inhibitor and suggests a possible link between IDE, UCP2, and amylin.

Support or Funding Information

National Science Foundation Research Initiation Award# 24430

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MAPK Signaling Influenced by Oxygen Environments
Authors: Forsberg Kersten , Chrestensen Carol
Published: June 2021

Mammalian cells thrive in a variety of different environments. In tissue culture there are many variables cells are exposed to; the impact of oxygen gas is one that is not well understood in relation to signaling pathways. In tissue culture, cells are commonly grown in atmospheric oxygen (~21%), yet the environment of many cells in vivo is considerably less (1% – 14% depending on the cells type). The increased oxygen content can cause higher levels of reactive oxide species (ROS). When ROS overwhelm the cell, normal cellular responses are affected. Extracellular kinase 1 and 2 (ERK 1/2) are proteins know to be directly modified by ROS, sometimes resulting in kinase inhibition. In this study we examined responses of the MAPK pathway to epidermal growth factor (EGF) in normoxic (6% O2) and atmospheric (21% O2) conditions. Baby hamster kidney (BHK‐21) or Human Dermal Epithelial (HMEC‐1) cells were grown and maintained in atmospheric conditions. The cells were then seeded and either kept under atmospheric conditions (normal tissue culture conditions) or moved to 6% oxygen in a Bactrox™(with all other conditions remaining constant). These cells were grown for two days until approximately 80–90% confluent and then they were serum starved and treated with EGF (0–30 minutes) in the two environments. They were harvested on ice in their respective environments. The lysates were then removed and frozen. After lysis the two conditions were treated in atmospheric oxygen. We hypothesized that the ERK 1/2 pathway responses would be different under the conditions of 6% versus 21% oxygen. ERK 1/2 phosphorylation status was assessed in parallel with kinase activity. Also explored was the difference in cell growth between the two environments. Our results show subtle differences in the peaks of phosphorylation and in the threshold of responses as well as differences in activity of the harvested ERK. Our work will lead to an enhanced understanding of how cellular signaling pathways are impacted by oxygen levels.

Support or Funding Information

NIH grant # R15 GM110634‐01A1 to C.A.C., and Kennesaw State University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Re‐polarization of tumor‐associated macrophages via reconstituted high‐density lipoprotein nanoparticles
Authors: Dossou Akpedje Serena , Sabnis Nirupama , Raut Sangram , Dasseux Jean‐Louis , Lacko Andras G.
Published: June 2021

Phenotypic plasticity of macrophages allows them to assume different functional profiles as they are called upon to respond to biological events. For example, the M1 phenotype of macrophage potentiates pro‐inflammatory and anti‐tumoral properties whereas the M2 phenotype is associated with immuno‐regulatory, tissue repair and pro‐tumoral properties. Tumor‐associated macrophages (TAMs) initially present anti‐tumoral behaviors via their “M1” phenotype. Under the stimulation of factors secreted by the malignant tumor, TAMs progressively take on a pro‐tumoral role via polarization to an “M2” phenotype. The M2 phenotype which supports immune‐dampening activities helps sustain tumor proliferation, immune system evasion, angiogenesis, tissue invasion and metastasis. TAMs, thus, make attractive targets for an anti‐cancer immunotherapy strategy. Considering the whole‐body distribution of macrophages, and their critical role in immune response, we propose to pursue the repolarization of M2‐type macrophages in the tumor microenvironment toward an M1 phenotype, utilizing a targeted drug delivery carrier.

Lipoproteins are natural transporters of hydrophobic substances in the blood, including lipids, hormones and drugs. Our lab developed a biocompatible reconstituted high‐density lipoprotein (rHDL) nanoparticle with properties highly similar to the native HDL. Dynamic light scattering analysis and transmission electron micrographs have confirmed a sub‐50 nm size for our particles and a spherical shape. In addition to siRNA molecules, hydrophobic drugs such as paclitaxel and valrubicin were successfully encapsulated by the rHDL nanoparticle. Photophysical studies showed that as a carrier, the rHDL holds its cargo at its core. Receptor blocking studies showed that scavenger receptor, class B type 1 (SR‐B1) expressed on the surface of the target cell mediate the intracellular delivery of the nanoparticle's payload by interacting with ApoA1, a major protein component of the nanoparticle. Also, protein expression analysis showed that M2‐type macrophages exhibit higher levels of SR‐B1 compared to M1‐type macrophages. The rHDL nanoparticles can transport hydrophobic small molecule inhibitors or non‐coding RNAs often used in subtype reversal/re‐polarization (M2 to M1) strategies. Most importantly, the SR‐B1 expression profile of M2‐type macrophage indicates that the rHDL is particularly suited for selective targeted delivery of these agents to TAMs.

Support or Funding Information

Cerenis Therapeutics

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The CH2 Domain of Pseudophosphatase MK‐STYX Regulates Neurite Outgrowth
Authors: Reed Kirstin , Zhang Ashley , Whitehurst Wesley , Hinton Shantá D.
Published: June 2021

Pseudoenzymes are emerging as key regulators in signal transduction cascades. More specifically, pseudophosphatases have been implicated in diseases such as Alzheimer's disease, cancer, metabolic disorder, and obesity, highlighting their importance in cellular processes. The unique catalytically inactive member of the mitogen‐activated protein kinase phosphatases (MKP) family, MK‐STYX (mitogen activated kinase phosphoserine/threonine/tyrosine binding protein), is a regulator in the stress response, apoptosis, and neuronal differentiation. We previously reported that MK‐STYX increases neurite outgrowths in PC‐12 cells through the RhoA signaling pathway. Recently, we found that MK‐STYX overexpressing cells produce more growth cones than control cells. Furthermore, immunoblots show that MK‐STYX overexpressing cells have increased actin expression, which is sustained in the presence or absence of nerve growth factor. These MK‐STYX‐induced neurites have post‐synaptic and pre‐synaptic properties when stained with the classical axon and dendrite markers, Tau‐1 and MAP2 (microtubule‐associated protein 2) respectively. Furthermore, a dopamine assay demonstrated that MK‐STYX‐induced neurites are dopaminergic. In primary neurons, MK‐STYX causes morphological changes, such as axons and dendrites being visibly indistinguishable. MK‐STYX also elicits a significant increase in the number of neurites per neuron in hippocampal primary neurons. Thus, it is apparent that MK‐STYX has a role in neuronal differentiation. To investigate the molecular mechanism that MK‐STYX utilizes to cause neurite formation, PC‐12 cells expressing GFP‐tagged truncated versions of MK‐STYX rhodanese (cdc 25 homology; CH2) or dual specificity phosphatase (DSP) domain and stimulated with NGF were analyzed for neurite outgrowths. Cells expressing the CH2 domain formed a significant number of neurites, whereas cells expressing only the DSP domain produced no neurites. Furthermore, cells expressing the DSP domain of MK‐STYX had significantly fewer neurites compared to cells expressing wild‐type MK‐STYX. Taken together, these data provide evidence that MK‐STYX is a regulator in neurite formation and that the CH2 domain is key for regulating neurite differentiation while the DSP domain may be the accessory domain. It also highlights that this unique member of the MKP subfamily potentially has a major role in neuronal signaling.

Support or Funding Information

Grace Blank Award; The Mary E. Ferguson Memorial Research Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PUFA supplementation may confer enhanced metabolic capacity in stimulated skeletal muscle cells
Authors: Symanietz Sarah , Conley Emily , Blair Jennifer , Petros Mera , Schlater Amber E
Published: June 2021

Skeletal muscle is an energetically demanding tissue that catabolizes a variety of different substrates, a phenomenon that is largely activity‐dependent. Lipids are an energetically‐rich macromolecule and, in this light, are advantageous for skeletal muscle performance and maintenance. It has long been established that lipids have dichotomous lasting effects on the body that appear to be, at least in part, species‐specific. While lipid‐related pathologies are largely associated with species such as saturated fatty acids, diacylglycerols, and ceramides, other lipid species, such as various unsaturated fatty acids, confer adaptive changes and may actually protect against diseases. Lipids have previously been linked to increased aerobic capacity in skeletal muscle, though the role of fatty acid quality in this context (e.g., degree of saturation) is less clear. In the present study, we used C2C12 cells, an immortalized line of mouse skeletal muscle cells, to better understand how polyunsaturated fatty acids (PUFAs), in combination with exercise, affect metabolism and protein concentration in skeletal muscle. Following standard proliferation protocols, mature myotubes were differentiated for 7 days in either control media or media supplemented with a 5% addition of linoleic acid (PUFA); additionally, to stimulate contractile activity, a subset of cell cohorts from each media group were exposed to caffeine for 20 minutes per day during differentiation days 4–7. All cells were harvested for protein on differentiation day seven. Protein concentration across cohorts was measured using a Bradford protein assay. Metabolic changes were characterized by running functional enzyme assays for lactate dehydrogenase (LDH) and citrate synthase (CS) to compare anaerobic and aerobic changes, respectively. Preliminary data show that non‐exercised cohorts have higher protein concentration when supplemented PUFA, and that exercise, both with and without PUFA supplementation, decreases protein concentration as compared to respective non‐exercised cohorts. Regarding enzyme activity, PUFA supplementation appears to decrease LDH activity in non‐exercised cells, while PUFA supplementation appears to increase LDH activity in exercised cells; CS activity also appears to change in PUFA supplemented cohorts. Collectively, these preliminary data suggest activity‐induced adaptive changes in skeletal muscle metabolism are contingent upon specific substrate availability, whereby PUFA availability may advantageously prime skeletal muscle for a higher overall metabolic capacity.

Support or Funding Information

McNair Scholars Program and The College of Saint Scholastica

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Generation of a tetracycline‐inducible NKCC2 expressing MDCKI cell line
Authors: Rosenbaek Lena Lindtoft , Rizzo Federica , Staub Olivier , Fenton Robert
Published: June 2021

In the kidney, the sodium chloride potassium cotransporter NKCC2 of the thick ascending limb (TAL) of the loop of Henle plays an important role in regulation of body salt and water homeostasis. Due to a lack of a suitable polarized epithelial cell line model, the majority of functional and cell biological studies of NKCC2 have been performed in isolated TAL tubules or following expression of NKCC2 in xenopus oocytes. The aim of this study was to generate a MDCKI cell line with stable and tetracycline‐inducible expression of NKCC2. Our previous studies using this approach have demonstrated that this is an excellent approach to study another member of the same protein family, the NaCl cotransporter NCC. FRT‐MDCKI cells transfected with the A‐isoform of human NKCC2 (hNKCC2‐A) had robust NKCC2 mRNA expression after 24 h of tetracycline induction. Enhanced NKCC2 protein expression was achieved by additional treatment of cells with the histone deacetylase inhibitor, valproic acid in conjunction with tetracycline. PNGase F treatment of cell lysates determined that NKCC2 was expressed as highly glycosylated monomers and dimers. NKCC2 was observed both intracellularly and at the apical pole of MDCKI‐hNKCC2‐A cells using immunofluorescence labeling and confocal microscopy. Cycloheximide chase studies determined that the mature form of NKCC2 has a half‐life of around 12 hours, whereas the immature, non‐glycosylated form of NKCC2‐A has a much shorter half‐life. NKCC2 levels in the apical membrane increased with forskolin or hypotonic low chloride stimulation, which were accompanied with significantly increased phosphorylation at Ser126 or Thr96, respectively. We conclude that the MDCKI‐hNKCC2 A cell line is an excellent model for studying NKCC2 function and regulation.

Support or Funding Information

DFF ‐ Danish Council for Independent Research, Medical Sciences

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fluorescence resonance energy transfer (FRET) based methods to study DNA topology and topoisomerases
Authors: Leng Fenfei
Published: June 2021

Previously, we reported the synthesis of a type of unique fluorescence‐labeled DNA molecules that can be used to study DNA topology and topoisomerases by fluorescence resonance energy transfer (FRET) (Gu et al, Scientific Reports, 2016, 6:36006). Specifically, we inserted an 82 nt. synthetic DNA oligomer FL905 or FL924 carrying a 42 nt. AT sequence with fluorescein and dabcyl or TAMRA and BHQ2 labels into a gapped DNA molecule to generate relaxed and supercoiled pAB1_FL905 or pAB1_FL924. Since the fluorescence intensity of pAB1_FL905 and pAB1_FL924 is dependent on its supercoiling status, these DNA molecules are powerful tools to study DNA topology and topoisomerases by FRET. Recently, we are able to use these unique plasmid DNA molecules to study kinetics of different DNA topoisomerases and find that all DNA topoisomerases follow the classical Michaelis‐Menten kinetics and have unique steady‐state kinetic parameters, KM, Vmax, and kcat. We also developed high throughput screening (HTS) assays to identify inhibitors/activators targeting different DNA topoisomerases including bacterial DNA gyrase and topoisomerase I.

Support or Funding Information

1R21AI125973‐01A1

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New Links Between the Autophagy Machinery and Cellular Homeostasis
Authors: Levine Beth , Liu Yang , Fernandez Alvaro Fernandez
Published: June 2021

The autophagy machinery is best‐known for its roles in targeting unwanted cargo for lysosomal degradation and other intracellular membrane trafficking events. In this talk, I will discuss our work identifying two novel nodes of molecular cross‐talk between the autophagy protein, Beclin 1, and cellular metabolism and homeostasis. Beclin 1 is a scaffold protein that regulates the lipid kinase activity of class III phosphatidylinositol 3‐kinase complexes that function in autophagy initiation and autophagosomal/endosomal maturation. Recently, we found that the function of Beclin 1 in cellular metabolism and homeostasis extends beyond autophagy and endocytosis. We identified an interaction between an innate immune sensor and Beclin 1 that regulates exercise‐induced activation of AMPK, a master regulator of cellular energy homeostasis, AMPK. We also identified a role for an interaction between Beclin 1 and the Na+,K+‐ATPase pump in the regulation of life and death decisions of the cell during tissue ischemic injury. Together, these findings suggest that autophagy proteins play a multifunctional role in cellular energy regulation and homeostasis.

Funding Sources:

National Institutes of Health

Cancer Prevention Research Institute of Texas

Fondation Leducq

Support or Funding Information

National Institutes of Health Cancer Prevention Research Institute of Texas Fondation Leducq

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hydralazine Improves Mitochondrial Function via SIRT1/SIRT5 Activation and Protects against Glucose Toxicity
Authors: Mirzaei Hamid Harrison , Dehgan Esmaeil
Published: June 2021

Mitochondrial dysfunction is one of the hallmarks of aging. Improving mitochondrial activity has been a prime target for improving organismal fitness and longevity and in many cases a potential treatment for age‐related diseases. In this study, we demonstrate that hydralazine, an FDA approved antihypertensive drug, improves mitochondrial function in a SIRT1/SIRT5‐dependent manner. We show that hydralazine treatment alters central carbon metabolism, increases TCA cycle metabolites, and improves respiration inC. elegans. Our data suggest that hydralazine‐induced mitochondrial activation is essential for the drug's prolongevity effect in C. elegans. We demonstrate that under glucose stress, hydralazine treatment restores locomotor performance, mitochondrial health, and lifespan in C. elegans. These new insights into hydralazine's ability to alter the mitochondrial activity and metabolic homeostasis deepen our understanding of hydralazine's mechanism of action and its potential health benefits and side effects.

Support or Funding Information

This work was supported by the National Institutes of Health (grant R03AG045504 to H.M.), Robert A. Welch Foundation (grant I‐1850 to H.M.) and the Cancer Prevention and Research Institute of Texas (grant R1121 to H.M.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crystal structures of the acyl transferases involved in the human cytosolic and mitochondrial fatty acid synthase
Authors: Misquitta Stephanie A. , Bunkoczi Gabor , Wu Xiaoqiu , Kavanagh Kathryn L. , Hozjan Viktoria , Oppermann Udo , Smith Stuart
Published: June 2021

The objective was to determine the structural basis for the specificities of acyl transferases involved in substrate loading from CoA thioester to the acyl carrier proteins of the type I cytosolic and type II mitochondrial fatty acid synthases. The type I enzyme (MAT) loads both the chain extender malonyl and acetyl primer substrate whereas the type II enzyme (MT) loads only malonyl moieties. Both transferases are characterized by a α/β ‐like hydrolase domain and a smaller domain with a ferredoxin‐like fold. The active‐site, located in a gorge between the two domains, is characterized by a serine nucleophile, supported by a histidine residue, an oxyanion hole and an arginine residue that anchors the 3‐carboxylate of the malonyl moiety. Replacement of the arginine residue severely compromises activity towards malonyl‐CoA but in MT induces activity toward acetyl‐CoA and in MAT induces activity toward acyl‐CoAs containing 2‐10 C‐atoms. Substrate docking experiments confirm the roles of the active site residues and reveal a cryptic acyl binding pocket in MAT that accommodates medium‐length acyl chains in mutants lacking the conserved arginine. Although the two enzymes share similar structural features, the MT more closely resembles its prokaryotic counterparts, consistent with the endosymbiotic theory of mitochondrial origin. Supported by NIH, DK16073 & GM069717. The SGC is supported by public and private sponsors.
Structure and Function of Human Senescence Marker Protein 30 (SMP30), Understanding the Zinc Dependent Gluconolactonase Activity and Calcium Regulation
Authors: Chakraborti Subhendu , Bahnson Brian J
Published: June 2021

Senescence Marker Protein 30 (SMP30) is a 34‐kDa protein whose expression in liver, kidney and lungs decreases, with aging. SMP30 has been identified as a zinc dependent gluconolactonase, which hydrolyses various aldonolactones. The lactonase reaction with L‐gulono‐γ‐lactone is the penultimate step in Vitamin‐C biosynthesis and SMP30 knockout mice are prone to scurvy. SMP30 is also known as regucalcin, which plays a major role in intracellular calcium homeostasis. There is no reported crystal structure of this enzyme. We believe that a structure will aid in elucidating the unresolved functions of SMP30. Human SMP30 was cloned, over‐expressed and pure active protein was crystallized. The crystals diffract to < 2.0 Angstrom. We have also obtained selenomethionine protein crystals, to solve the phases. We are performing group specific labeling and site‐directed mutagenesis in order to characterize the active site. The crystal structure will complement this approach. Enzymatic assays of gluconolactonase have shown interesting metal ion behavior. Although the highest activity was observed with Zn2+, we have also observed activity with Ca2+. The use of chelating agents like EGTA/EDTA, dyes like Quin‐2, Terbium binding assays, CD spectroscopy and ITC will further characterize the role of Ca2+ in SMP30 structure and function.

In the long term, our studies will reveal its overall physiological function.
Homocysteine stimulates chemokine expression in the kidney via NF‐kappa B activation
Authors: Hwang Sun‐Young , Siow Yaw L. , Karmin O.
Published: June 2021

Hyperhomocysteinemia, a condition of elevated blood homocysteine levels, is a risk factor for cardiovascular disorders. Renal disease is an important factor causing hyperhomocysteinemia, the direct effect of homocysteine on the kidney is not well documented. One of the important features in kidney diseases is the infiltration of monocyte/macrophage in the kidney. Monocyte chemoattractant protein‐1 (MCP‐1) is a potent chemokine that stimulates monocyte migration into the tissue. The aim of this study was to investigate the effect of hyperhomocysteinemia on MCP‐1 expression and the underling mechanisms in rat kidneys. Hyperhomocysteinemia was induced in rats fed a high‐methionine diet. The nuclear factor kappa‐B (NF‐κB) activity, the levels of MCP‐1 mRNA and protein were significantly increased in the kidneys of hyperhomocysteinemic rats. Pretreatment of hyperhomocysteinemic rats with a NF‐κB inhibitor completely abolished hyperhomocysteinemia‐induced MCP‐1 expression in the kidney. This further confirmed the causative role of NF‐κB activation in hyperhomocysteinemia‐induced MCP‐1 expression. Taken together, these results suggest that diet‐induced hyperhomocysteinemia can stimulate chemokine expression in the kidney via NF‐κB activation. Such an inflammatory response may contribute to renal injury and chronic systemic inflammation associated with hyperhomocysteinemia.
Structural Comparison of the TM Regions of HIV‐1, Gp41 and Potential Viral Co‐Receptors CD4 and Syndecan
Authors: Feltzin Andy , Bell Ellis
Published: June 2021

Using Clustal and Blast analysis in conjunction with site directed mutagenesis we identified a region of HIV gp41 essential to the infectious fusion capabilities of the virus. The TM region is highly conserved in HIV‐1 and chimpanzee SIV, but not HIV‐2, and is shared by human CD4 [CD4 is a cell surface receptor that facilitates binding interactions and has a particularly significant role in immune cells; T‐cells and phagocytes] and by Syndecan [a heparin sulfate proteoglycan involved in receptor mediated cell migration and chemotaxis], both known coreceptors of HIV‐1. This region has not previously been identified in these coreceptors as a potentially significant region. Using a combination of direct experimentation [2D NMR in SDS micelles] and computational modeling [computational alanine scanning, GRASP analysis of the electrostatic surfaces and surface area analysis] it is clear that these TM regions share a common structural motif and could provide for an interaction surface either for homo oligomerization or for hetero‐oligomerization. Computational docking studies favor models involving hetero‐dimerization. This raises the possibility that the co‐receptor function of CD4 or Syndecan may involve interactions with gp41 within the plane of the cell membrane as well as extracellular interactions, and contribute to the mechanism of entry of the virus into target cells.
SACCHAROMYCES CEREVISIAE HEP1 PROMOTES THE SOLUBILITY OF NON‐COGNATE CHAPERONES AND REQUIRES BOUND ZINC FOR SOLUBILITY
Authors: Zhai Peng , Stanworth Crystal , Nguyen Peter , Liu Shirley , Silberg Jonathan
Published: June 2021

In yeast, Hsp70 escort protein Hep1 regulates the solubility and functions of mitochondrial molecular chaperones, which are essential for protein folding, protein translocation, and Fe/S‐cluster biogenesis. Little is known about why escort proteins have evolved to regulate chaperone functions and whether human mitochondrial Hsp70 (mtHsp70) also requires escort proteins. We have examined the metal content, stability, and substrate specificity of Saccharomyces cerevisiae Hep1 (ScHep1). Elemental analysis revealed that Hep1 contains stoichiometric levels of zinc, and mutation of any one of its four conserved cysteines to serine abolished Hep1 solubility upon expression in Escherichia coli. In vitro analysis of Hep1 solubility further showed that removal of zinc by the strong oxidant methanethiolsulphonate led to aggregation, whereas zinc‐bound Hep1 remained soluble when heated from 20 to 70°C and exhibited little change in its circular dichroism spectrum. A possible interaction between yeast Hep1 and human mtHsp70 was also examined by measuring the influence of Hep1 on the solubility of mtHsp70. When expressed alone in Escherichia coli, mtHsp70 was insoluble. However, coexpression with Hep1 enhanced the level of soluble mtHsp70 produced. Furthermore, gel filtration analysis revealed that mtHsp70 self‐associates in the absence of nucleotides, and dissociates into monomers when incubated with Hep1 or ATP. Ongoing work is investigating whether human Hep has similar in vivo and in vitro functions as yeast Hep. These findings provide evidence that Hep1 requires bound zinc to maintain its solubility, and they implicate a role for escort proteins in regulating the oligomerization of human chaperones. They also suggest that Hep homologs may adjust chaperone solubility and function in response to biological oxidants.
Thrombin mediated regulation of CCN1 regulates cell proliferation in an integrin dependent manner
Authors: Walsh Colin T. , Hsiao Albert , Stupack Dwayne , Brown Joan Heller
Published: June 2021

We previously reported that PAR1 (activated by thrombin) and the M3 muscarinic (activated by carbachol) receptors differentially effect AP‐1 mediated gene expression and DNA synthesis in 1321N1 astroglial cells. Microarray experiments were used to explore genes that were differently regulated by thrombin verses carbachol and revealed that the secreted protein cysteine‐rich 61 (Cyr61/CCN1) was robustly and selectively induced by thrombin. Thrombin but not carbachol activate RhoA therefore we postulated that Rho signaling mediates CCN1 expression. This was supported by experiments demonstrating that the C3 exoenzyme attenuates thrombin‐induced CCN1 expression and that other GPCR agonists that activate Rho also induce CCN1 expression while Gq coupled receptor agonists do not. Inhibition of CCN1 expression with siRNA decreased thrombin‐stimulated DNA synthesis by ~50% implicating CCN1 signaling in thrombin mediated proliferation. Currently integrins are the only known receptor for CCN1. Data shows that CCN1 is present on the cell surface of 1321N1 cells, that 1321N1 cells bind to CCN1 in an α6β1, α5β1, and β1 integrin dependent manner, and that functional inhibitory antibodies to these integrins attenuate thrombin mediated DNA synthesis. We suggest that CCN1 represents an intermediate by which G12/13 and Rho coupled GPCRs utilize integrins to elicit additional signals involved in cell proliferation.

NIH 5R01‐GM36927
Characterization of novel neurotensin‐like peptides from the neural complex of the ascidian, Ciona intestinalis
Authors: Kawada Tsuyoshi , Itoh Yoshiyuki , Ogasawara Michio , Sekiguchi Toshio , Satake Honoo
Published: June 2021

The ascidian, Ciona intestinalis, has a pivotal evolutionary position as a protochordate. Comprehensive analyses of the Ciona neural complex extracts using mass spectrometry followed by the Ciona genomic DNA/EST database‐searching detected more than novel twenty peptides including six neurotensin‐homologous or ‐analogous peptides encoded in two genes. Neurotensin is a brain/gut peptide involved in hypothermia, antinociceptin, dopamine neurotransmission, and stimulation of anterior pituitary hormone secretion as a neuropeptide in the central nervous system, and digestive and cardiovascular functions as a paracrine and endocrine factor in peripheral tissues. The neurotensin family peptides were identified in only mammals and birds but not from other vertebrate species. The Ciona neurotensin‐like peptides contain the PSIL, PSII or PSVI sequences at their C‐termini, which are analogous to the neurotensin consensus sequence RRPYIL responsible for their physiological actions. In this presentation, we show the primary sequences gene organization and localization of the Ciona neurotensin‐like peptides. Our results suggest that the ancestral neurotensin gene is conserved in Ciona intestinalis.
Spatiotemporal regulation of HSH2 expression is critical for the humoral immune response.
Authors: King Rodney Glenn , Justement Louis
Published: June 2021

The adaptor protein HSH2 has been shown to be dynamically expressed in cells of the B lineage. HSH2 is upregulated in response to TLR agonists and stimulation through the TNF family receptors CD40 and BAFF‐R in vitro. In mice challenged with T‐dependent antigen, HSH2 is expressed exclusively in the light zone of germinal centers. To address the function of HSH2, transgenic mice were generated resulting in the constitutive expression of HSH2 in all cells of the B lineage. These mice display a mild B cell developmental defect manifest by a delay in population of the peripheral B cell pool and reduction of late transitional populations. BrdU labeling demonstrated a reduced efficiency of B cell maturation in the periphery; however the B cell compartment of adult HSH2 Tg mice is relatively normal. The serum Ig titers of HSH2 Tg mice, however, are reduced in comparison to wild type. In addition, HSH2 Tg animals exhibited a significant impairment in their response to immunization with both T‐independent and T‐dependent antigens. B cells isolated from HSH2 Tg animals fail to respond normally to stimulation through BAFF‐R and CD40 receptors although their response to TLR agonists is unaffected. These data suggest that regulation of HSH2 expression is critical for normal B cell responses to signals delivered via receptors of the TNF family thereby controlling the humoral immune response.
The Trafficking of Viral Immune Evasion Protein m4/gp34 and MHC class I complexes is Facilitated by Another Viral Gene m168 in Murine Cytomegalovirus Infections
Authors: Lu Xiuju , Cho Kathy , Hill Ann
Published: June 2021

The murine cytomegalovirus (MCMV) encodes immune evasion protein m4/gp34, which is unique among known viral genes that target MHC class I pathway of antigen presentation. It associates with MHC class I molecules at the cell surface, however, it inhibits antigen recognition without reducing cell surface class I levels. We have found that the maturation of m4/gp34 and associated MHC class I molecule Kb was impaired when m4 was expressed in a gutless adenovector Ad‐m4. The current study defined more clearly the necessity and sufficiency of MCMV environment in facilitating the export of m4‐Kb complexes and identified the MCMV gene that functions to affect the complexes transport: 1) The retention of m4/gp34‐Kb complexes in Ad‐m4 infection was not caused by differences of endogenous m4/gp34 over‐expression, nor by the differences of m4/gp34 synthetic rate between Ad‐m4 and MCMV infections; 2) Super‐infection of Ad‐m4 with m4‐deletant MCMV mutant recovers the maturation of m4‐Kb complexes, which convinced that the trafficking of m4/gp34‐Kb complexes was manipulated by MCMV; 3) MCMV gene m168 was identified facilitating the export of m4/gp34‐Kb complexes. Further study is focusing on the biological meaning of m168 in promoting m4/gp34‐Kb complexes export, and the molecular mechanisms of how m168 facilitates the export of m4/gp34‐Kb complexes.
N‐terminal trimer extension of nominal CD8 T cell epitopes is sufficient to promote cross‐presentation to cognate CD8 T cells in vivo
Authors: Wei Cheng‐Hong , Sherman Linda A
Published: June 2021

Cross‐presentation pathway is essential for generating effective CD8 T cell responses against cell‐associated antigens derived from either infectious pathogens or malignant cells. In this study, we investigated the regulation of cross‐presentation pathway through manipulation of the CD8 T cell epitopes using TCR transgenic mice as model system. Previous studies have shown that nominal peptide is inefficiently cross‐presented and that proteins and large polypeptides that require proteasomal processing are the main source of naturally cross‐presented antigens. Here we show that N‐terminal extension of nominal peptide by as few as three residues is sufficient to produce a substrate for TAP dependent cross‐presentation that is highly efficient in cross‐priming murine CD8 T cells in vivo. On a molar basis, cross‐priming with trimer extended peptide is 20 fold more efficient than priming with intact protein. Depending on the presence or absence of inflammatory signals, this method can lead to immunity or tolerance. Moreover, cross‐presentation of N‐terminal modified CD8 epitopes derived from insulin can significantly protect NOD mice from developing type I diabetes. Therefore, this method of peptide extension should prove of great value in facilitating in vivo studies of CD8 immunity and tolerance that rely on cross‐presentation.

(This work was supported by NIH grants DK50824 and U19 AI050864)
Signaling via LTβR & HVEM‐BTLA Pathways Control Dendritic Cell Homeostasis In Vivo
Authors: De Trez Carl , Schneider Kirsten , Ha Sukwon , Benedict Chris A. , Ware Carl F.
Published: June 2021

Proliferation of dendritic cells (DC) in the spleen is regulated by positive growth signals through the Lymphotoxin (LT)‐β receptor, however the countering inhibitory signals that achieve homeostatic control are unresolved. Mice deficient in LTβR and the ligands, LTα or LTβ, but not LIGHT as well as in NF‐κB inducing kinase (NIK) show a specific loss of CD8α‐ DC subsets. In contrast, the CD8α‐ DC subsets were overpopulated in mice deficient in the herpesvirus entry mediator (HVEM) or B and T lymphocyte attenuator (BTLA). HVEM and BTLA DC subsets displayed a specific growth advantage in repopulating the spleen in competitive replacement bone marrow chimeric mice. Expression of HVEM and BTLA was required in DC and in the surrounding microenvironment, although DC expression of LTβR was necessary to maintain homeostasis. Moreover, enforced activation of LTβR with an agonist antibody drove expansion of CD8α‐ DC subsets overriding the HVEM‐BTLA checkpoint. These results indicate the HVEM‐BTLA pathway provides an inhibitory checkpoint regulating DC homeostasis in lymphoid tissue. Together, the LTβR and HVEM‐BTLA pathways form an integrated signaling network regulating DC homeostasis.