bluuebunny/crossref_metadata_2025 huggingface dataset:
Prion formation in yeast is influenced by alterations of the ubiquitin proteolysis
Authors: Chernova Tatiana A. , Allen Kim D. , Chernoff Yury O. , Wilkinson Keith D.
Published: June 2021

Prions are infectious aggregated proteins that cause neurodegenerative diseases in mammals and transmit heritable traits in yeast. De novo appearance of a yeast prion is facilitated by an increase in the concentration of the same protein or by the presence of aggregates of a different QN‐rich protein. Since levels of protein in the cell are controlled at least in part by ubiquitin proteasome system (UPS), we investigated the role of UPS in maintenance of a prion isoform of the yeast translation termination factor Sup35. Deletion of the ubiquitin‐conjugating enzyme Ubc4 increased de novo prion formation and antagonized prion elimination by the disaggregating chaperone Hsp104. Although levels of Sup35 protein were not affected by this UPS alteration, levels of the QN‐rich protein Pin3 were increased. We confirmed that overexpression of Pin3 protein stimulates prion formation by Sup35 and demonstrated that Pin3 is present in the cell in the ubiquitinated form. Prevention of Pin3 ubiquitination by mutation has an effect on protein stability, cellular localization and aggregation. Our data suggest that UPS can regulate prion formation via modulation of QN‐rich proteins in the yeast model.(Supported by grant GM30308 from NIH)
Mapping the site of inositol 1,4,5‐trisphosphate receptor ubiquitination
Authors: Sliter Danielle , Kirkpatrick Donald , Alzayady Kamil , Gygi Stephen
Published: June 2021

Inositol 1,4,5‐trisphosphate (IP3) receptors form tetrameric channels in endoplasmic reticulum (ER) membranes, and mediate IP3‐induced calcium mobilization in mammalian cells. In response to various extracellular stimuli that persistently increase phospholipase C activity, IP3 receptors are ubiquitinated and then degraded by the 26S proteasome. We have previously shown that IP3 receptor ubiquitination is triggered by the combined action of IP3 and calcium, is mediated by the ubiquitin‐conjugating enzyme, mamUbc7, and is processed by the ER‐associated degradation pathway. Here, for endogenous type 1 IP3 receptor processed by endogenous enzymes, we sought to determine the sites of ubiquitination and the composition of attached ubiquitin chains. Using mass spectrometry we found that a total 43 of the 167 lysines in the receptor are ubiquitinated, with three lysines, K1771, K1884, and K1901, being most frequently identified. Additionally, using ubiquitin‐AQUA analysis we found that most of these sites (~85%) are occupied by monoubiquitin and that both K48 and K63 linkages are abundant in attached chains. Overall, these data show that an endogenous substrate ubiquitinated in situ by endogenous ligases is tagged widely with an array of ubiquitin species.

This research was funded by a grant to RJH Wojcikiewicz by the National Institues of Health.
Deficiency of Tyk2 kinase in mice leads to obesity and insulin resistance
Authors: Gornicka Agnieszka Olga , Millward Carrie , Croniger Colleen , Larner Andrew
Published: June 2021

The prevalence of obesity and type 2 diabetes has reached epidemic proportions worldwide. Tyk2 is one of the Janus (JAK) kinases that are stimulated by several cytokines, IL‐12, IL‐10, IL‐18. The JAK/STAT pathway can be also activated by leptin and indirectly by insulin. To study the role of Tyk2 in the development of obesity and diabetes, we analyzed mice with a gene‐targeted deletion of Tyk2 kinase (Tyk2 −/− mice). Tyk2−/− mice and wild type (WT) mice were fed a chow diet (17% fat) for 3 and 12 months. The animals gained similar weight at 3 months of age, but by 12 months of age, the Tyk2 −/− mice had increased body weight (40.8 %) compared to WT mice. The food intake in both Tyk2 −/− and WT mice was very similar suggesting alterations in overall energy expenditure. To understand the underlying mechanism, we analyzed expression of genes involved in lipogenesis and overall energy metabolism. Both 3 month‐old and 12 month‐old Tyk2 −/− had very low expression of uncoupling proteins 1 and 3 (UCP‐1 and UCP‐3) in muscle, brown and white adipose tissue compared to WT mice. Plasma levels of insulin, leptin, triglycerides and cholesterol were elevated in 12 months old Tyk2 −/− mice. The Tyk2−/− mice had impaired glucose tolerance tests and they developed steatosis by 12 months. The Tyk2 −/− mice had increased expression of hepatic lipogenic genes, acetyl CoA carboxylase (ACC) and fatty acid synthase (FAS). Interestingly, levels of β‐oxidation genes in brown adipose tissue and muscle were reduced in both 3 moths old and 12 months old Tyk2 −/− mice and may contribute to the underlying mechanism for the development of obesity. The above data indicate that Tyk2−/− mice develop obesity and type 2 diabetes as they age and thus Tyk2 kinase plays an important role in the development of these diseases.
Increase of Glucose‐6‐Phosphate Dehydrogenase in Macrophage Is Associated with Enhanced Inflammatory Responses in Obesity
Authors: Ham Mira , Lee Joo‐Won , Choi A Hyun , Masuzaki Hiroaki , Kim Jae Bum
Published: June 2021

The role of Glucose‐6‐phosphate dehydrogenase(G6PD), NADPH producing enzyme, has been received a great attention in metabolic disorder such as obesity, diabetes and atherosclerosis. Previously, we have demonstrated that increased expression of G6PD in adipose tissue of obese animals provokes insulin resistance and stimulates inflammatory response and ROS production in adipocytes. To further understand the role of G6PD in metabolic disorders, we investigated G6PD in macrophages. Interestingly, the level of G6PD expression in stromal vascular fraction of adipose tissue was increased in obese animals, which exhibit enhanced infiltrating monocyte and macrophages. Additionally, the expression of G6PD in macrophage was stimulated by inflammatory signals such as LPS. Moreover, overexpression of G6PD in macrophage stimulated the expression of inflammatory genes including MCP‐1, IL‐6 and iNOS, implying that enhanced G6PD activity is associated with augmentation of inflammatory response in adipose tissue of obese subjects. Together, it is plausible to speculate that abnormal increase of G6PD in macrophage would modulate expression profile of pro‐inflammatory cytokines, which could deteriorate whole body inflammatory signaling in obese subjects.

This work was supported in part by grants from the Molecular and Cellular BioDiscovery Research Program (M10748000258‐07N4800‐25810) and the National Research Laboratory Program (R0A‐2004‐000‐10359‐0) of Korea Science and Engineering Foundation (KOSEF). J.B.K. and M.H. was supported by a BK21 Research Fellowship from the Ministry of Education and Human Resources Development.
New pyranine receptor for use in endovesiculation assay
Authors: Wood Ryan Matthew , Sidorov Vladimir
Published: June 2021

The endovesiculation assay is a method for determining the amount of external surroundings that have been internalized into large unilamellar vesicles (LUVs). In our assay, internalization of the fluorescent 8‐hydroxypyrene‐1,3,6‐trisulfonate (HPTS, pyranine) is used to quantify the extent of endovesiculation. After endovesiculation is induced, a receptor for HPTS is applied. The fluorescence of the external dye is quenched by the receptor, and the residual fluorescence corresponds to the amount of dye internalized by endovesiculation.

The amount of HPTS dye internalized in the assay is low, and a receptor that is highly sensitive is needed. The dynamic quencher p‐xylene‐bis‐pyridinium bromide (DPX) provides the necessary sensitivity because of its 99.7% quench of HPTS fluorescence.1 However, DPX requires 40 mM concentrations that do not mimic biological conditions. A class of cyclen‐based receptors offers a better alternative because they operate at significantly lower concentrations.2 Of the cyclen‐based derivatives that we have synthesized, nitrophenyl‐urea‐cyclen has the highest fluorescence quench (97.6%) and can be applied to the endovesiculation assay.
Mass spectrometry of oxidized peptides from peroxisomal malate synthase and catalase
Authors: Anand Pria , Donaldson Robert P , Simha Rahul
Published: June 2021

Oxidative damage is irreparable and marks proteins for degradation. If oxidized proteins are not degraded, they can form aggregates linked to age‐related disorders. In peroxisomes such damage is likely due to the generation of reactive oxygen species. Our purpose was to observe the effects of oxidation on peroxisomal proteins. Peroxisomal proteins were subjected to H2O2 in vivo, then analyzed for oxidation using Western blotting with a biotin/avidin detection system. Mass spectrometry was used along with a novel JAVA program to identify oxidized peptides within catalase (CAT) and malate synthase (MS). H2O2 exposure was found to increase the activity of CAT, and to increase the number of oxidized peptides found in both CAT and MS. When an oxidized protein was tagged with biotin hydrazide, several peptides were observed less frequently in the mass spectra than in the non‐oxidized sample. Biotin tagged peptides shift to different masses. CAT had 13 peptides that were affected by oxidation and MS had 10. These peptides have definable locations within the proteins. This type of analysis could lead to an understanding of how sites of oxidation relate to functional alterations, degradation or aggregation. NSF REU #0649165
Large scale purification of recombinant bovine deoxyhypusine hydroxylase
Authors: Smith Joshua L , Wang Yinglu , Huang Jenq‐Kuen , Wen Lisa
Published: June 2021

The mature eukaryotic initiation factor 5A (eIF5A) is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine. Hypusine synthesis is a posttranslational modification catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) which is essential for the function of eIF5A in cell proliferation and survival. It has been suggested that posttranslational modifications of eIF5A could be a suitable target for anticancer therapy. A detailed knowledge of structure‐function relationship of either enzyme will help in designing better inhibitors thus better control of proliferative diseases.

Recently, we have cloned and expressed functional bovine DOHH (bDOHH) (Huang et al., Protein Expr. Purif. 54, 126–133, 2007). Here we report large scale purification of bDOHH. The GST‐tagged DOHH fusion protein was overexpressed by IPTG induction and the fusion protein purified by affinity chromatography with a glutathione agarose column. The GST tag was removed by thrombin digestion. The bDOHH protein was then separated from the GST carrier by glutathione agarose. The monomic bDOHH protein was separated from aggregates by gel filtration on a Superdex‐200 column. The purified protein is being used for initial crystallization screening.

Supported by grants from National Institutes of Health 1R15 GM60266‐01A1 and the University Research Council at WIU.
Purification of MBP‐tagged proteins using new prepacked columns
Authors: Karlsson Anneli Birgit Marianne , Bergh Ann , Busson Karolina , Carlsson Marianne , Heijbel Anna , Lundqvist Jon , Maier Esther , Zimmerman Rhyenne , Zou Jinyu
Published: June 2021

The success of protein production and subsequent analysis are dependent on the expression levels, solubility and purification of the protein. Maltose Binding Protein (MBP) is often used as an affinity fusion tag. Several studies have shown that MBP‐tagged proteins have high solubility compared to other tagged proteins.

The new MBPTrap™ HP consists of an affinity medium, Dextrin Sepharose™ High Performance, packed in 1‐ml and 5‐ml HiTrap™ columns. The medium has high specificity and capacity. Additionally, it can withstand harsh Cleaning In Place (CIP) procedures, like sodium hydroxide, without losing binding capacity. The HiTrap format enables automated protocols to be used on ÄKTAdesign™ systems as well as with syringe or laboratory pump.

In this work, MBP‐tagged proteins from E. coli were purified on prepacked 1‐ml or 5‐ml MBPTrap HP columns. The purity of the eluted target proteins was greater than 90 %. A stability study was performed, six purification runs each followed by a CIP run (0.5 M NaOH) were made on the same column. The results show no significant change in yield or purity. The results of an automated two step purification on ÄKTAxpress™ is shown. The affinity chromatography step on MBPTrap HP was followed by a gel filtration step.

In summary, the results show that the prepacked MBPTrap HP column has high binding capacity and the eluted target protein is very pure. Furthermore, the medium is stable in 0.5 M NaOH.
Mapping and Characterizing aura, a Cytokinesis Mutant
Authors: Solanki Bharti Teresa , Pelegri Francisco
Published: June 2021

A fundamental biological phenomenon during embryogenesis in animal cells is the process of cell division, an event that divides daughter cells in two. The completion of cytokinesis often results in the asymmetric segregation of cell determinants, thereby allowing the two daughter cells to proceed towards diverse cell fates. Many of these processes are driven and regulated by maternal products already present in the unfertilized egg. A recessive maternal‐effect mutation, aura, was identified in a zebrafish gynogenesis‐based screen. Preliminary evidence indicates that aura affects several events during early development that may be dependent on microtubule cytoskeletal rearrangements. aura mutants exhibit defects in cortical granule exocytosis, the recruitment of cell adhesion components to the furrow, germ plasm segregation and late cytokinesis. A positional cloning approach was used to determine the location and identity of aura. A lesion in a candidate gene has been found and rescue efforts are now underway to confirm the identity of the gene. Cell biology, genetic, and experimental approaches are being used to characterize aura gene function further. Together, these studies will contribute to understanding the function and regulation of aura and its role in the cytokinesis pathway.

Sources of funding: NIH Ruth L. Kirschstein NRSA MARC Fellowhship and NIH R01 Grant.
Probing the influence of ligand binding on cytochrome P450 enzyme remodeling and dynamics: Using molecular dynamics simulation to give insight into multiple cytochrome 2B4 structures
Authors: Shahrokh Kiumars , Yost Garold S. , Cheatham Thomas E.
Published: June 2021

Hepatic P450 enzymes metabolize a broad range of endogenous and exogenous substrates. Despite the emergence of both substrate‐free and substrate‐bound P450 structures, the structural rearrangements for the accommodation of such a wide range substrates by these enzymes are not yet predictable. To better understand the implications of substrate binding on the active site structure and protein dynamics we have performed a series of 100 ns‐scale molecular dynamics (MD) simulations on three different crystal structures of the 2B4 isozyme. These include a single ligand‐free monomeric 2B4 in an “open” structure from the crystallographic dimer (PDB code: ), 2B4 bound to a small ligand: 4‐(4‐chlorophenyl) imidazole (PDB code: ), and 2B4 bound with a larger ligand: bifonazole (PDB code: ). For each structure, molecular dynamics simulations using modern force fields and simulation protocols were applied. Additional simulations probed the full set of variants that swap substrates or do not include substrate, to assess how the enzymes may remodel in the presence, absence, or with a change of substrate. The MD simulations provide detailed insight into the structure and dynamics of the 2B4 enzymes with their substrates, and show the protein adaptation to substrate binding. Supported by NIH grant # GM074249, NIH grant # NCRR 1 S10 RR17214‐01 and the University of Utah Center for High Performance Computing.
CDK5: A Contributor to the Insulin Phenotype of Transdifferentiated β Cells
Authors: Stevens Jessica Lee , Aguanno Ann
Published: June 2021

We investigate the role of CDK5 in mammalian development. Although CDK5 belongs to the cyclin dependent kinase family of proteins involved in regulating the cell cycle, CDK5 itself has been shown to influence differentiation of non‐dividing nervous tissues. Previously our lab has used PC12 cells to study CDK5's role in neuronal development. Experiments involving growth factor (NGF, FGF) induced differentiation of PC12 cells in the presence of a CDK5 inhibitor resulted in drastic morphological changes. Recent reports suggest CDK5 may also play a extra neuronal role. Therefore, we have explored the impact of CDK5 inhibition in pancreatic tissue using the acinar cell line AR42J as our model. We have investigated insulin production in the presence or absence of CDK5. To achieve this goal, the cells were differentiated into insulin‐producing cells by the administration of the chemical exendin‐4. CDK5 activity was inhibited by exposure to olomoucine, and the two flavonoids, kampherol and quercetin. Our results suggest that CDK5 plays a role in both the secretion and production of insulin.

This research was supported by the Rose M. Badgley Charitable Trust, The Murray Foundation and the Division of Sciences, Marymount Manhattan College.
Nde1/Ndel1 are required to load dynein onto microtubules in Xenopus egg extracts
Authors: Kijanska Monika Anna , Zylkiewicz Eliza , Choi Won‐Chan , Derewenda Zygmunt S. , Stukenberg P. Todd
Published: June 2021

Dynein is the minus‐end directed microtubule motor, required for membrane transport, nuclear and neuronal migration and regulation of the mitotic spindle. To perform so many functions, the loading of dynein onto microtubules must be tightly regulated. Nde1 and Ndel1 are closely related proteins that have been implicated in a number of dynein related processes. Nde1 and Ndel1 are coiled‐coil proteins with the central Lis1 interacting domain and the highly phosphorylated C‐terminus that interacts with regulatory proteins.

Addition of constitutively active Ran to Xenopus laevis egg extract nucleates microtubules that become substrate for motors and form asters with the minus ends of microtubules bundled in a central focus. We find extracts depleted of Nde1 and Ndel1 are unable to form asters. The addition of Ndel1 lacking the C‐terminal domain can rescue these extracts, suggesting that the C‐terminal domain is not essential for aster formation. Surprisingly, point mutants in the Lis1 interaction domain also partially rescue, indicating that Ndel1 may have Lis1 independent functions. Dynein and dynactin were not loaded onto microtubules in the absence of Nde1/Ndel1. This is surprising because in a purified system dynein/dynactin load onto microtubules independently of cofactors. However our data show that in a complex system dynein loading requires Nde1/Ndel1 function.

Supported by NIH grant NS 036267.
Fatty acids regulate TNF‐α production by J774 cells
Authors: Thais Martins de Lima , Alice Cristina Rodrigues , Camargo Rocha Michelle , Curi Rui
Published: June 2021

Chronic inflammation has been reported in obesity‐related insulin resistance. Most of the adipose tissue‐derived cytokines, including TNF‐α, are originated from the infiltrated macrophage population. Macrophage accumulation has been associated to the increase in adiposity. However, molecular signals that regulate macrophage function in obese adipose tissue are not well known. The effect of various fatty acids (FA) on TNF‐α production by J774 cells (a model of murine macrophages) treated with 2.5μg/mL LPS was evaluated. Palmitic, stearic, oleic, linoleic, arachidonic, DHA and EPA acids were added at different concentrations 3 hours prior to LPS stimulation for 24 hours. Palmitic, stearic, DHA and EPA at 50μM, but not lower concentrations, increased LPS stimulatory effect on TNF‐α production. In the absence of LPS, all FA, with exception of oleic and linoleic acids, stimulated TNF‐α production, although TNF‐α concentrations in the medium were extremely lower. Real‐time PCR analysis showed that the stimulatory effect of FA on LPS induced TNF‐α expression occurred between 3 and 6 hours of treatment. NF‐κB activation was also increased after all FA treatments. These findings indicate that saturated and omega‐3 FA present similar effects on TNF‐α production by macrophages, via NF‐κB activation. Financial support: FAPESP, CNPq.
Conserved C‐terminal arginines are crucial for targeting PNPLA5/GS2‐like triglyceride lipase to lipid droplets
Authors: Murugesan Sricharan , Goldberg Elysa B. , Brown William J.
Published: June 2021

Lipid droplets (LDs) are highly conserved storage depots for triglycerides (TGs) and cholesterol esters. The Patatin‐like Phospholipase Domain containing protein A (PNPLA1‐9) family of neutral lipases play key roles in the hydrolysis of stored TGs and in the regulation of LD metabolism. Previous work in our lab has uncovered a conserved charge‐dependent LD targeting motif (LTM) in the C‐terminal third of human PNPLA5 and Brummer Lipase (BL), the Drosophila homolog of human Adipose Triglyceride Lipase (PNPLA2/ATGL). The LTM of PNPLA5 is a conserved basic patch containing three arginine residues, two of which, if mutated to alanines abolish LD association. Here we report that each of these arginine residues is crucial for targeting PNPLA5 to LDs. Transient transfection of full‐length PNPLA5 containing all possible arginine to alanine changes revealed that altering any one of the arginines abolished LD association in vivo. However, mutations of similar putative basic patches in human PNPLA2/ATGL revealed only mild mislocalization. These results show that although conserved arginines in basic patches of PNPLA5 and BL identify a conserved LTM, not all family members are recruited to LDs in an identical manner. This correlates with evidence that PNPLA family members serve overlapping, but not identical, roles in TG homeostasis.

This work supported by Hatch Project NYC‐165415 and NIH grant DK51596 to WJB.
FATP1 and FATP4 mediate long‐chain fatty acid‐induced activation of AMP‐activated protein kinase in adipocytes
Authors: Wiczer Brian Michael , Lobo Sandra , Bernlohr David A
Published: June 2021

Fatty acid transport protein 1 (FATP1) and FATP4 are the two main FATP family members expressed in adipocytes and are integral membrane proteins that exhibit both long‐chain and very long‐chain acyl‐CoA synthetase activity. The ATP‐dependent acyl‐CoA synthetase reaction of the FATPs has been difficult to study due to the inherent instability of purified enzyme in detergent micelles. To circumvent this problem both FATP1 and FATP4 were reconstituted unidirectionally in an inside‐out orientation into small unilamellar vesicles and the catalytic activity assessed. Reconstituted FATP1 and FATP4 exhibited a specific activity 50% that of the protein in detergent micelles and was independent of the lipid vesicle composition. Importantly, the thermostability of reconstituted FATPs was increased 30‐fold compared with that in detergent micelles. This increased stability allowed for the demonstration that AMP is produced as a product of the FATP acyl‐CoA synthetase reaction. Consistent with AMP production, palmitate influx into 3T3‐L1 adipocytes induced AMP‐activated protein kinase (AMPK) activation, acetyl‐CoA carboxylase phosphorylation, and was decreased in cell lines expressing shRNA targeting either FATP1 or FATP4. These results demonstrate that fatty acid influx and storage, mediated by FATP1 or FATP4 catalytic activity, regulate the AMP‐activated protein kinase. Research supported by ADA‐RA‐12.
Investigating Human and Drosophila Sprinter Function
Authors: Harmon Andrew William , Selva Erica M
Published: June 2021

Within developing tissues, the Wingless (Wg)/Wnt signaling pathway acts as a morphogen, regulating downstream target activation within receiving cells and ensuring proper growth and organization. Our laboratory has identified a novel highly conserved transmembrane protein in Drosophila named Sprinter (Srt) whose specific mechanism of function is unknown, however without it, Wg is retained within secretory cells (1). We hypothesize that Srt functions by supplying a critical activity required for maturation of Wg protein in its route through the secretory pathway. The focus of this research is to begin to characterize Srt function and determine if the mechanism of function is conserved between humans and Drosophila. Compartment localization studies show that the majority of Drosophila and human Srt reside in an undefined secretory compartment and high level Srt expression causes cells to produce processes that appear to contain vesicles of Wg (Figure 1). Furthermore, we have found that Srt exerts its function by direct physical interaction with the Wg protein and that this interaction is conserved across species. These results suggest that Srt is a multifunctional protein that physically interacts with Wg and perhaps promotes cellular morphological changes that support its dissemination from secretory cells. This data also indicates that these functions are conserved across species. Furthermore, these experiments provide the foundation for evaluating Srt function and conservation in developing Drosophila.
PAR1‐mediated stable platelet aggregation requires temporal regulation of Rap1 activity by phosphatidylinositol phosphates (PIPns).
Authors: Holinstat Michael , Hudson W. James , Preininger Anita M. , Milne Stephen B. , Brown H. Alex , Hamm Heidi E.
Published: June 2021

Thrombin is the most potent activator of platelets through reversible and then irreversible aggregation and it mediates a diverse range of vascular effects which may result in acute coronary syndrome and stroke. We demonstrate that PAR1, but not PAR4, signals human platelet activation through generation of a phosphatidylinositol phosphate (PIPn) signaling pathway which mediates Rap1 regulation of stable platelet activation. To investigate this signaling requirement for PIPns, we examined the activation kinetics of Rap1, platelet aggregation, protein complex formation and proteomics. Treatment with PI‐kinase inhibitors significantly altered the kinetics of PAR1‐, but not PAR4‐mediated Rap1 activation and platelet aggregation. Additionally, PI5K and PI3Kγ were required for stable PAR1‐mediated platelet aggregation and Rap1 was found to form a protein complex with both PI3K and PI5K following PAR1 stimulation. Although stable platelet activation is sensitive to perturbation of the PI‐kinase pathway, early Rap1 activation and platelet aggregation are insensitive to perturbations along the PI‐kinase pathway. Hence, this novel lipid signaling pathway is a potential target for anti‐platelet therapy. This study was supported in part by grants P50 HL081009 (to H.E.H.) and K99 HL089457 (to M.H.) from the National Institutes of Health.
Comparison of caspase‐8 fluorescent substrates for development of microtiter plate homogeneous cell‐based assay
Authors: Rakhmanova Vera , Han Xing , Tong Xiaohe , Hong Anita L. , Meyer Rich
Published: June 2021

Caspase‐8 is an initiator enzyme accepting signals from death receptors and activating downstream effectors caspases (caspase‐3, ‐7) through proteolytic cleavage. Caspase‐8 has substrate specificity for the amino acid sequence, Ile‐Glu‐Thr‐Asp (IETD). Although there are a number of assays using IETD based caspase‐8 substrates, those protocols are not optimized for 96‐well plate format and include additional steps of cell harvesting, lysate preparation and subsequent aliquoting of prepared lysates into 96‐well plates.

To develop homogeneous 96‐well plate caspase‐8 fluorimetric assays, we compared a series of fluorophores (AMC, AFC and Rh110) linked to IETD peptide. AMC and AFC‐linked substrates tested with purified enzyme gave at least a 2‐fold higher signal/background ratio and better kinetic parameters than the Rh110 labeled substrate. When applied to cells grown and lysed in 96‐well plates, the Ac‐IETD‐AMC substrate showed a high background. The longer‐wavelength spectra of the AFC (Ex/Em=380 nm/500 nm) provided greater sensitivity and less interference from cell media. The use of Ac‐IETD‐AFC substrate for detection of caspase‐8 activity in cells enabled the development of a homogeneous assay with a wide linear range and high signal/background ratio. This assay is ideal for measuring enzyme activity in cells grown in 96‐well plates and for screening of apoptosis inducers or inhibitors.
CD45 Phosphatase Domain Required For Regulation Of Galectin‐1 Induced T cell Death
Authors: Earl Lesley , Bi Shuguang , Baum Linda G.
Published: June 2021

CD45, a T cell surface glycoprotein, regulates susceptibility to galectin‐1 (gal‐1) cell death. Differential glycosylation allows CD45 to positively or negatively regulate the gal‐1 death signal. Modification of CD45 with core 2 O‐glycans, by core 2 O‐GlcNAc transferase (C2GnT), enhances susceptibility to gal‐1, while absence of core 2 O‐glycans on CD45 reduces susceptibility to gal‐1. T cells expressing C2GnT, with core 2 O‐glycans on CD45, cluster CD45 on the cell surface and have reduced CD45 phosphatase activity after gal‐1 binding; gal‐1 resistance of T cells lacking core 2 O‐glycans can be overcome by inhibition of phosphatase activity. To understand how the CD45 phosphatase controls susceptibility to gal‐1, we expressed a series of CD45 constructs with various mutations in the intracellular domain in T cells with and without C2GnT. We find that enhancement of gal‐1 susceptibility by core 2 O‐glycans requires the CD45 intracellular domain. Thus, binding of gal‐1 to specific glycans on the CD45 extracellular domain regulates intracellular phosphatase activity. We have made a series of gal‐1 analogues that vary orientation and spacing of the carbohydrate recognition domains (CRDs) of gal‐1 and found that increasing spacing of the CRDs increases the potency of the gal‐1 death signal. The effect of gal‐1 CRD spacing on regulation of CD45 phosphatase activity will be discussed.
Tolfenamic Acid Increases EGR‐1 Expression through a PKC/ERK Dependent Pathway in Human Colorectal Cancer Cells
Authors: Lee Seong‐Ho , Whitlock Nichelle C , Choi Chang Kyoung , English Anthony E , Safe Stephen H , Baek Seung J
Published: June 2021

Considerable evidence indicates that the use of non‐steroidal anti‐inflammatory drugs (NSAIDs) reduces the mortality of colorectal cancer in a cyclooxygenase (COX)‐independent manner. Tolfenamic acid (TA) has been selected as a potent chemopreventive agent for cancer therapy because of its inhibitory effect on tumorigenesis in pancreatic cancer models. In the present study, we sought to the anti‐cancer effect of TA in human colorectal cancer. The mechanisms by which TA affects anti‐tumorigenesis involve the induction of the proapoptotic protein EGR‐1 in human colorectal cancer cells. TA treatment inhibited cell proliferation and induced apoptosis in HCT‐116 cells. Electrical impedance study showed that TA resulted in a decrease of the normalized impedance components which reflect increasing numbers of apoptotic cells. Ectopic expression of EGR‐1 increased apoptosis, and siRNA of EGR‐1 reduced TA‐induced apoptosis. TA treatment induced approximately 5.5‐fold induction of luciferase activity of EGR‐1 promoter. Subsequently, the results of the luciferase assay using internal deletion clones showed that the EBS and CREB/ATF binding sites in the EGR‐1 promoter are required for TA‐induced EGR‐1 transactivation. Experiments using kinase inhibitors and siRNA transfection implied the involvement of the PKC/ERK pathway in TA‐induced EGR‐1 expression. These results demonstrate a role of EGR‐1 in TA‐induced anti‐tumorigenesis in human colorectal cancer cells.
Expanding the Scope of Small Molecule Transcription Activators
Authors: Casey Ryan James , Desaulniers Jean‐Paul , Leathen Mathew L , Hojfeldt Jonas W , Mapp Anna K
Published: June 2021

Mis‐regulated transcription correlates strongly with many human disease states ranging from cancer to diabetes. Synthetic molecules that can directly perturb gene expression patterns thus hold great therapeutic potential. However, therapeutically useful activators that up‐regulate transcription have yet to be described. To this end, we have developed small molecules based on an isoxazolidine scaffold to serve as transcriptional activation domain (TAD) replacements. Such molecules can up‐regulate transcription in living cells when localized to DNA at an EC50 of 33 nM and the maximal activity of 80‐fold activation occurs at 1 μM. These molecules can be used to probe activator and co activator function which provides valuable insight into activator recruitment of transcription factors. Specifically, these molecules can inhibit activator co‐activator interactions and provide insight into how activators bind their targets. Through structure‐activity based studies, we identified key residues important for transcription and establish a relationship between hydrophobicity and activation. Further, to confirm the importance of a structured scaffold, we synthesized a peptide with the similar functionality of our most important isoxazolidine and transcription activation was attenuated. These conclusions lead us to believe that activators should be well structured and have a blend of hydrophobicity and polar functionality. The NSF (CAREER), the American Cancer Society (RSG 05‐195‐01) and the Sloan Foundation funded this research.
The glycogenolysosome: A protein interaction network determined by students in an undergraduate hypothesis‐driven cell biology laboratory course
Authors: Rice Nancy A.
Published: June 2021

At Western Kentucky University, we use a research‐oriented approach to teach the cell biology laboratory (Biol 412). The course is divided into two projects organized in an independent learning and discovery environment. One of these projects is a 10‐week long yeast two‐hybrid screen. Over the past three years, students have screened two proteins known to regulate glycogenolysis, glycogen phosphorylase (GPb) and phosporylase kinase (PhK), against a rabbit skeletal muscle cDNA library to search for protein‐protein interactions. From these experiments, students have identified several novel associations. Specifically, using two different constructs of GPb as bait, the regulatory domain (a.a. 17‐484) or the catalytic domain (a.a. 485–843), students identified muscle creatine kinase (M‐CK) and phosphoglycerate mutase as binding partners. As a validation for this screen, one student group, using the regulatory domain as bait, identified a positive association with GPb that corresponded to the C‐terminus of this protein. When the β subunit of PhK was used as bait, a positive association was identified also with M‐CK, as well as skeletal muscle α‐actinin, actin, and the α subunit of PhK (residues 424–579). The identification of PhK α was again a good positive control for the experiment. The identification of M‐CK as a binding partner with both PhK and GPb suggests that the CK pathway may serve as the primary source of ATP production for these enzymes during periods of high energy demand. Thus, by using various baits over successive years in the Biol 412 course, we have assembled a protein interaction network which we termed the “glycogenolysosome”. Moreover, this course has taught students basic cell and molecular biology techniques in an exciting research‐based format while instructing them in proper experimental design and data analysis.
Building High Efficiency Learning Teams in the Biochemistry Laboratory and Classroom
Authors: Stahelin Robert Virgil
Published: June 2021

Learning for entering the culture and practice of science takes place in three program‐embedded‐areas: laboratory‐centered, program centered, and peer‐centered. Additionally, group‐based work brings together skills at acquiring, communicating, and integrating information, while prospective employers support programs that allow the growth of interpersonal, organizational, and teamwork skills that will better prepare students for the scientific workforce. Team Based Learning (TBL) originated with Larry Michaelsen in the late 1970's. TBL is an especially powerful form of learning as it creates learning teams, which are distinct from other group learning activities. Based upon our preliminary studies and the current state of literature in the education field, we hypothesized that high efficiency learning teams could be created in the academic laboratory and undergraduate/graduate biochemistry classroom through implementation of TBL. First, a new course addressing the biophysics of lipids has been designed and implemented. Second, mentoring of undergraduate and graduate research teams performing research in the Stahelin lab utilizes TBL for efficient scientific discovery. The outline, assessment, advantages and disadvantages, as well as the future of TBL in a bona fide scientific research setting and classroom will be presented.
Introducing Students to the Process of Science – through Modeling
Authors: Herman Tim , Colton Shannon , Franzen Margaret , Nelson David , Keck James
Published: June 2021

The MSOE Center for BioMolecular Modeling (CBM) is an instructional materials development lab focused on creating physical protein models by rapid prototyping. Protein images created in a computer setting can be built as physical models with this technology. For 6 years the CBM has trained secondary science teachers in model design. SMART Teams (Students Modeling A Research Topic) have evolved from this program. They consist of a trained teacher and 4–5 of their students. After a training phase, the team is matched with a local research lab. The team visits the lab to learn about a structure‐based research project, then designs and builds a physical model of the protein. The program culminates with a poster and presentation. This outreach program is an effective, minimally invasive mechanism to introduce students to the “real world of science” in active research labs. By involving graduate students and postdocs as SMART Team mentors, we expose future faculty to principles of Scientific Teaching, in which the process of science is emphasized over the facts. We will describe our current efforts to disseminate SMART Teams and a related program – Proteins in Active Learning Modules – in which we work with educators to create physical models and supporting visualizations to teach difficult concepts to undergraduates. This work is supported by grants from NIH‐NCRR SEPA, NSF‐CCLI and HHMI Precollege Science Education Program.
The Immune System: Defenders of Our Health
Authors: Withum Elizabeth Anne , Mcloughlin Rachel
Published: June 2021

The Immune system is one of many specialized systems of the body. This system has developed over millions of years to identify, label and protect us from foreign pathogens such as bacteria, virus, parasites and protozoans. These lessons are designed for middle school as an introduction to the human bodies Immune System and how we detect that the immune system is there and working properly. A specific focus will be on the neutrophil as the defender against bacterial attack.

A modified ELISA has been developed using a commercially available ELISA kit purchased from an educational biological supplier. The ELISA will be used to detect chemokine levels in a mock patient infection sample. Students will interpret the ELISA results to establish chemokine levels in their patient samples. Finally, students will make the connection between chemokine levels, neutrophil recruitment and bacterial clearance that is necessary for a ealthy immune response.
mTORC2 in T Lymphocyte Development and Proliferation
Authors: LEE KEUNWOOK , Cho Sung Hoon , Potter Ross M , Gudapati Prathyusha , Park Do‐Sim , Magnuson Mark A , Killeen Nigel , Boothby Mark
Published: June 2021

PDK1 and its downstream target Akt contribute both to pre‐TCR and TCR signal transduction important for proliferation and survival of thymocytes undergoing β‐selection or mature T cells. Mammalian target of rapamycin (mTOR) controls various cell functions and is a target of immunosuppressant drugs. Recent biochemical studies suggest that mTOR complex 2 (mTORC2) may be the exclusive kinase for phosphorylation of S473 in Akt. However, the role of mTORC2 in T lineage is not known. Here, we show that mTORC2 regulates T cell development and cytokine‐induced proliferation. To assess the consequences of reduced activity of mTORC2 in T lymphocytes, we generated mice with T lineage‐specific depletion of rictor, an indispensible component of mTORC2. The results indicate that mTORC2 is required for normal DN/DP transition and suggest a role in positive selection process. Furthermore, mTORC2 haplo‐insufficiency in peripheral T cells impairs cell cycle progression induced by cytokines whose receptor share the common γ chain without affecting survival. Overall, these findings indicate that mTORC2 is a crucial signal relay in T cell development and homeostasis, presumably by regulating phosphorylation status of Akt.
The Nucleotide‐binding Oligomerisation Domain Protein 1 in T cells Modulates TCR Mediated Responses in the Thymus and Periphery
Authors: Li Xiangli , Sarvetnick Nora E
Published: June 2021

The Nucleotide‐binding oligomerisation domain protein 1 (NOD1), which functions as an intracellular sensor of bacteria, has been shown to have roles in both innate and adaptive immune responses. However, it is still unknown that T cell expression of NOD1 impacts T cell development and activation. Here we demonstrate that T cell expression of NOD1 restricts the sensitivity of T cells to TCR triggering and participates in the intra‐thymic differentiation of T cells and their proliferation in the periphery. When NOD1 is absent there is an increased commitment to the CD4 T cell lineage in the thymus and a skewed CD4 vs CD8 T cell ratios in the periphery, a phenomenon more notable after infection with either virus or bacteria. In addition, NOD1 deficient CD8 T cells exhibit increased proliferation and activation in response to antigen challenge in vitro and in vivo. Our study supports an immunoregulatory role for NOD1 expressed in T cells in the development and maintenance of a fit adaptive immune system.
Functional characterization of human T cells generated in vitro from hematopoietic stem cells
Authors: Awong Geneve , Sajic Rade , Motte‐Mohs Ross La , Cochrane Alan , Zúñiga‐Pflücker Juan Carlos
Published: June 2021

T lymphocytes are a class of white blood cells that play a major role in the adaptive immune response. T cells are generated in the unique microenvironment of the thymus; however, the precise molecular signals enabling their differentiation are still being elucidated. Using the OP9‐DL1 stromal system we have demonstrated the generation of human T cells from umbilical cord‐blood (UCB) hematopoietic stem cells (HSCs). Here, we characterize T cell specification and commitment events induced at both the phenotypic and molecular level examined by the acquisition of T‐lineage cell surface markers and the induction of T cell associated genes, respectively. In addition, in vitro derived T cell subsets are mature and functional as they proliferate and secrete IFNγ following stimulation. Furthermore, we demonstrate the ability of in vitro‐derived human T cell progenitors to engraft and differentiate into a mouse host thymus in vitro. Finally, HSCs transduced to express an HIV (Human Immunodeficiency virus) inhibitory construct have been generated, and will be evaluated for decreased susceptibility to infection when challenged with HIV. This system has important clinical implications as it provides the foundation for a large supply of human T lymphocyte progenitors, which could be used for the immune‐reconstitution of T cell‐depleted individuals.

Research supported by the Ontario HIV Treatment Network (OHTN) and the Canadian Institutes of Health Research (CIHR)
Molecular mechanism of allelic exclusion of TCR alpha chains
Authors: Rybakin Vasily , Gascoigne Nicholas R. J.
Published: June 2021

Allelic exclusion is a mechanism whereby a product of single allele is selected for expression. In case of the T cell receptor, allelic exclusion takes place differently for TCRa and TCRb chains. For the TCRb chain, a successfully completed in‐frame rearrangement of one locus effectively inhibits the rearrangement of the second locus. This inhibition is achieved as a result of signaling originated from a successfully formed dimer of the b chain with pre‐Ta.

In contrast, both TCRa alleles are rearranged, often multiple times, until the positive selection stage, when a single a chain in conjunction with a fixed b chain allows the thymocyte to differentiate. Majority of immature thymocytes express more than one a chain on the cell surface, while after positive selection only one chain is expressed.

We developed and experimental system allowing to directly assess the fate of each of two specific a chains by flow cytometry and microscopy. Our studies are aimed at the better characterization of posttranslational modifications and signaling events leading to the selection of a single TCRa chain expressed at the cell surface.
Upregulation of CD56 expression and T‐cell receptor (TCR)‐independent activation of T cells with aging
Authors: Lemster Bonnie H , Michel Joshua J , Montag David T , Paat John , Studenski Stephanie A , Newman Anne B , Vallejo Abbe N
Published: June 2021

Degeneration of the thymus and severe contraction of the T‐cell repertoire with aging suggest that immune homeostasis in old age could be mediated by distinct effectors. Therefore we examined receptors expressed on T cells as they undergo senescence in vitro, and those displayed by circulating T cells during normal chronologic aging. Monitoring of T cells driven to senescence showed de novo induction of CD56, the prototypic receptor of NK cells. Analysis of fresh T cells in blood showed an age‐dependent induction of CD56. These unusual T cells expressed high levels of Bcl2, p16, and p53, and had limited, or completely lost, ability to undergo cell division; properties consistent with senescence. CD56 cross‐linking without TCR ligation on CD56+ T cells resulted in extensive protein phosphorylation, NFκB activation, and Bax down‐regulation. CD56 cross‐linking was also sufficient to drive production of various humoral factors. These data indicate that the immunologic environment in old age is functionally distinct, rather than a dysfunctional version of that seen at a young age. In view of the reported inefficiencies of TCR‐driven immunity with aging, elicitation of TCR‐independent immune cascades by CD56+ T cells provide new avenues for the development of alternative strategies to enhance protective immunity in the elderly. [Supported by NIH grants R01‐AG022379, R01‐AG023629, P20‐CA103730, and T35‐AG026778].
Role of PARP‐14 in B cell survival signaling
Authors: Cho Sung Hoon , Goenka Shreevrat , Gudapati Prathyusha , Henttinen Tiina , Reinikainen Arja , Lahesmaa Riitta , Boothby Mark
Published: June 2021

Poly‐(ADP‐Ribose) Polymerases (PARPs) catalyze formation of branched ADP‐ribose polymers by transfer of ADP‐ribose residues from NAD+ to target protein. PARPs are involved in a wide range of biological processes including inflammation, regulation of gene expression, and DNA damage repair. Although there are 17 PARP superfamily members, very little is known about their function except that of PARP‐1. PARP‐14, a member of macrodomain‐containing PARP subfamily, is highly expressed in lymphoid organs and interacts with the IL‐4‐activated transcription factor Stat6. To elucidate physiological functions of PARP‐14, we have generated and analyzed PARP‐14 deficient mice. IL‐4 was unable to enhance the proliferation of anti‐IgM‐stimulated PARP‐14 KO B cells, and the Ag‐specific IgA response was reduced in mice lacking this enzyme. In addition, PARP‐14 mediates IL‐4‐induced protection against apoptosis following irradiation or growth factor withdrawal, and regulates IL‐4 effect on the levels of gene products related to cell survival. Collectively, the results establish that PARP‐14 plays a crucial role in IL‐4 regulation of gene expression and lymphocyte functions.