bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional and Structural Insights into RSC‐mediated Nucleosome Remodeling via In‐Vivo Crosslinking
Authors: Evans Brian , Wilkins Bryan
Published: June 2021

In eukaryotes, genetic information is stored as chromatin. Within the chromatin, DNA is wound around octameric units of histone proteins. The histone‐DNA complex forms the nucleosome and represents the most basic level of DNA compaction. A class of proteins known as chromatin remodelers physically reposition nucleosomes along the chromatin fiber. This can directly affect and mediate many important processes such as DNA replication, repair, and transcription. Albeit, due to their large sizes and numerous auxiliary subunits, chromatin remodeler interactions at the nucleosome have yet to be fully characterized and remain relatively obscure. The RSC complex is the most abundant remodeling complex of budding yeast, with homologs in both animals and plants. In this work, we use synthetic biology to trap histone‐RSC remodeler interactions in living cells using the unnatural amino acid p‐benzoylphenylalanine (pBPA). We site‐specifically insert pBPA into histones and identify remodeler binding via the addition of short peptide fusion tags to our interaction target protein. This system has the potential to illuminate chromatin remodeler complex interactions which can then be used to identify and assign biologically important structures. By properly implementing the system, we exemplify the potential of this technology for creating crosslinking maps for RSC. This conclusion is supported by Western blot analysis and a clearly identified crosslink between a myc‐tagged subunit of RSC (Sth1 protein) with histone H2A and histone H3. In addition, we show that binding to H3 (but not H2A) is dependent upon PTMs and a defined acetylation event at lysine 14 on histone H3. In cells that lack H3 K14ac, Sth1 can no longer be sequestered to the nucleosome providing in vivo mechanistic details about RSC‐nucleosomal function/structure relationships.

Support or Funding Information

NIH R15 Academic Research Enhancement Award (AREA)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NLRP3 Inflammasome regulation of lipolysis in aged adipose tissue
Authors: Camell Christina , Dixit Vishwa Deep
Published: June 2021

Visceral adiposity is increased in the elderly and this increase is associated with increased risk for metabolic diseases. During aging, there are also increases in inflammation, senescence and leukocyte alterations in white adipose tissue. Furthermore, catecholamine‐induced lipolysis, the first step in generation of energy substrates through hydrolysis of triglycerides (TGs), declines with age. The defect in mobilization of free fatty acids (FFA) in elderly is accompanied with increased visceral adiposity, lower exercise capacity, failure to maintain core body temperature during cold stress, and reduced ability to survive starvation. While catecholamine signaling in adipocytes is normal in elderly, how lipolysis is impaired in aging remains unknown. Here we show that unlike diet‐induced obesity, aging is associated with reduction in adipose tissue macrophages (ATMs). We found ATMs regulate age‐related reduction in adipocyte lipolysis by lowering the bioavailability of norepinephrine (NE). Unbiased whole transcriptome analyses of ATMs revealed that aging upregulates genes controlling catecholamine degradation in an NLRP3 inflammasome‐dependent manner. Deletion of NLRP3 inflammasome in aging restored catecholamine‐induced lipolysis through downregulation of monoamine oxidase‐a (MAOA) that is known to degrade NE. Furthermore, inhibition of MAOA reversed age‐related reduction in adipose tissue NE concentration and restored lipolysis with increased levels of key lipolytic enzymes, adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL). Overall our findings suggest that NLRP3 inflammasome‐dependent immune‐metabolic interactions within AT contribute to age‐related inflammation and metabolic dysfunction. Our study also reveals that targeting neuro‐innate signaling between sympathetic nervous system and macrophages may offer new approaches to mitigate chronic inflammation‐induced metabolic impairment and functional decline.

Support or Funding Information

This work was funded in part by a supplemental award from the National Institute of Aging (grant number: 3R01AG043608‐03S1 FAIN:R01AG043608), the Irene Diamond AFAR Postdoctoral Transition Award, and K99AG058800

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression and purification of N‐terminally acetylated Ssa1 chaperone from Saccharomyces cerevisiae
Authors: Griffith Alijah A. , Holmes William
Published: June 2021

N‐terminal acetylation leads to diversity of a given organism's proteome through constituting a cell‐wide acetylome wherein polypeptides exhibit an altered function and dynamic, acting as a basis for cell signaling pathways and allowing a given cell to react specifically to environmental stressors or cellular perturbations. The reaction of N‐terminal acetylation is catalyzed by a collection of N‐terminal acetyltransferases (NATs), wherein NAT complexes transfer acetyl groups from acetyl‐coenzyme A to the a‐amino group at amino‐terminal residues, effectively neutralizing the positive charge of the amino residue. One potential target of N‐terminal acetylation is thought to be molecular chaperone Ssa1. Endogenous to the eukaryotic Saccharomyces cerevisiae, Ssa1 is HSP70 class molecular chaperone that works in tandem with its co‐chaperone Sis1 to maintain cellular proteostasis through restoring native conformations of misfolded proteins through a transient mechanism dictated by ATP hydrolysis. Overall, the consequences of N‐terminal acetylation on the Ssa1 function and ability to maintain cellular proteostasis remains to be fully characterized. To further probe the functional and mechanistic changes associated with the N‐terminal acetylation of Ssa1, we developed an efficient, user‐friendly, one‐step purification of Ssa1 utilizing a previously constructed Protein‐A transformant strain. Our results indicate that our method is able to natively isolate high yields of pure Ssa1 from small amounts of starting material in a short period of time while allowing for the native elution of Ssa1 without compromising enzymatic activity, allowing the eluted protein to be utilized in downstream analyses to better understand interactions between Ssa1 and its co‐chaperones as well as the effects of post‐translational modifications on chaperone function.

Support or Funding Information

Research supported by RI‐INBRE (#P20GM103430) from NIGMS, NIH.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Methionine sulfoxide reductase reverses oxidation of the nucleotide exchange factor Fes1 to regulate cytoplasmic Hsp70 chaperone cycle
Authors: Nicklow Erin E , Kim Jinnie , Sevier Carolyn S
Published: June 2021

Methionine residues in proteins are targets of oxidation by reactive oxygen species (ROS). Selective oxidation of methionine to methionine sulfoxide (MetO) can allow for reversible, beneficial modification of proteins to limit cell damage during conditions of excess ROS. Methionine sulfoxide reductases (Msrs) are conserved enzymes that reduce MetO residues in proteins, serving to both limit the over‐oxidation of protein methionines, and to remove MetO adducts when stress subsides. Although loss of Msr function has been implicated in neurodegenerative disorders and aging, it is unclear how Msr activities influence these disease pathologies because the vast majority of their physiological substrates remain unknown.

We have identified the cytoplasmic Hsp70 nucleotide exchange factor Fes1 as an Msr substrate in yeast. Under oxidative stress, Fes1 is oxidized at a cluster of N‐terminal methionine residues. Current data show that this Msr‐controlled modification reversibly regulates interaction between Fes1 and Hsp70, thereby modulating chaperone nucleotide exchange, ATP hydrolysis, and peptide binding and release. We suggest that these altered Hsp70 activities, as a consequence of Fes1 modification, may help cells cope with elevated ROS by limiting peptide aggregation and/or activating cellular stress response pathways, whereas sustained modification of Fes1 in the absence of Msr activity is detrimental to proteostasis under non‐stress conditions. Moreover, our studies with Fes1 uncovered that the two yeast Msr proteins (Mxr1 and Mxr2) are expressed in the cytoplasm, in contrast to prior studies of Mxr2 suggesting sole mitochondrial localization. We show that Mxr1 and Mxr2 co‐operate to regulate the redox state of Fes1 both in vivo and in vitro. Together, these studies provide a new perspective on how activities of the well‐studied Hsp70 family can be modulated by ROS, and further define how methionine modification is used as a means for cells to sense and respond to oxidative stress.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Auto‐proteolysis of Type 1 Metacaspases in Schizophyllum commune
Authors: Cummings Brianna Paige , Fox Kristin M.
Published: June 2021

Apoptosis is a specialized form of programmed cell that occurs under conditions such as stress, aging, and development. A key marker of apoptosis is the presence of caspases that cleave peptide bonds after aspartate residues via a catalytic histidine/cystine dyad. A group of related enzymes called metacaspases are found in plants, protozoa and fungi. Like caspases, they contain an active site histidine/cystine dyad and have a similar protein fold, yet they show preference for lysine and arginine residues and many require calcium for cleavage. Furthermore, Type I metacaspases contain a proline‐rich N‐terminal prodomain that is typically cleaved to achieve the fully active form. In some Type I metacaspases, continued auto‐proteolysis is observed after the prodomain is removed.

Within the fungus Schizophyllum commune genome five type I metacaspases (ScMC1‐5) have been characterized and expressed with and without the prodomain. Additionally, optimal pH and calcium concentrations have been determined for each of these metacaspases. To further understand the auto‐proteolysis of these metacaspases, attention was focused on ScMC1. A time course has been established over which ScMC1, in the presence of calcium, auto‐processes to form a 40kDa fragment. Fluorescent activity assays over the same time course have shown a decrease in overall activity on small peptide substrates of the incubated enzyme. Additionally, the H211Q/C267A active site double mutant form of ScMC1 showed decreased activity and no auto‐proteolysis upon incubation with calcium. Lastly, incubation of wild‐type ScMC2‐4 with the double mutant ScMC1 showed no proteolysis of ScMC1, indicating cleavage of ScMC1 by other Type I metacaspases does not occur. These results have furthered our understanding of metacaspase activation and function, and ultimately their role in fungal apoptosis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Integration of signaling pathways and physical forces during vascular growth
Authors: Iruela‐Arispe Luisa , Aragon Raquel , McDonald Austin
Published: June 2021

Our understanding of vascular morphogenesis and angiogenic growth has progressed considerably during the last few decades. Today the field has clarified the framework of interacting signaling pathways, intracellular regulatory genes and participating physical forces involved in the process of endothelial cell sprouting, lumen formation, and vascular remodeling. Nonetheless, once formed, vascular tubes also expand in width and length within the context of shear stress and tensional forces. Currently little information is available on how blood vessels perform this task or even what is the average half‐life of endothelial cells in the vascular wall. Using a combination of EdU incorporation, genetic tracing, rigorous quantification, and SILAM proteomics, our laboratory has gathered substantial information that sheds light into the cellular mechanisms and molecular players involved. The questions that will be addressed in this talk include: How does a vessel like the aorta expand in length and width while exposed to multiple physical forces? Do endothelial cells in an adult vessel divide to renew the endothelial lining or do [some] endothelial cells last for the lifetime of an individual? If endothelial cells proliferate: Is there a particular site/niche or do all cells all have equivalent capacity? Do endothelial cells have a finite number of cell divisions? What is the half‐life of endothelial cells in adult, normal blood vessels? What are the regulatory mechanisms involved in promoting vascular expansion? Taken together, we view this information as fundamental to our understanding the mechanisms controlling endothelial regeneration and their deregulations in settings like chronic/acute inflammation, aging, chronic diseases and physical trauma.

Support or Funding Information

NIH/NHLBI R35HL140014

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovering novel enzymes and metabolic pathways
Authors: Gerlt John
Published: June 2021

The number of proteins in the UniProt database (>127M sequences in Release 2018_08) is increasing with a doubling time of 2.5 yrs; however, ≥50% of the proteins have uncertain, unknown, or incorrect functions. The lecture will describe publicly accessible (“democratized”) web tools for protein sequence similarity networks (SNNs; EFI‐EST; efi.igb.illinois.edu/efi‐est/), 2) genome neighborhood networks (GNNs; EFI‐GNT; efi.igb.illinois.edu/efi‐gnt/), and 3) chemically guided functional profiling (EFI‐CGFP). These tools are used to 1) analyze sequence‐function space in protein families (EFI‐EST), 2) identify functionally linked enzymes in metabolic pathways (EFI‐GNT), and 3) prioritize protein families for functional assignment based on metagenome abundance (EFI‐CGFP). Examples will be described of the use of SSNs, GNNs, and CGFP to discover novel metabolic pathways in diverse microbial species, including the human gut microbiome.

Support or Funding Information

NIH/NIGMS P01GM118303

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EFFECT OF PHOTOBIOMODULATION ON MUSCLE REPAIR AND OXIDATIVE STRESS CAUSED BY BOTHROPS JARARACUSSU VENOM
Authors: Silva Luciana Miato Gonçalves , Freitas Sarah Cristina Ferreira , Zamuner Luis Fernando , Cogo José Carlos , d'Angelis Katia , Zamuner Stella Regina
Published: June 2021

INTRODUCTION

Envenoming caused by snakes of the genus Bothrops is often associated with severe and complex local pathological manifestations, with dermonecrosis and myonecrosis being the most severe damage. The specific treatment for Bothrops accident is the use of the antivenom, with the function to neutralize the circulating venom, but its action does not extend the local manifestations. Previous studies in our group have shown that photobiomodulation using low intensity laser (LBI) increases the viability of C2C12 muscle cells when incubated with B. jararacussu venom (BjV) and further promotes the proliferation of these cells.

AIM

to analyze the effect of LBI on muscle repair and the oxidative stress caused by B. jararacussu venom on C2C12 myoblast cells.

METHODOLOGY

Myoblasts cells were plated at 1×104 cells/well and incubated for 24 hours for cell adhesion. BjV (12.5ug/ml) was applied and the cells were immediately irradiated with LBI (660 and 780 nm, power of 100 mW, irradiated time 10 sec, energy density of 4 J/cm2) and incubated for 1 h. Then, a wound was created by scratching cells with a sterile pipette tip and the cell migration into the wound surface was analyzed 24 h after the venom incubation. Oxidative stress (NO, H2O2, SOD, Gpx and TBARS) were analyzed 1 h after BjV incubation. The NO concentration was evaluated by the GRIESS and the H2O2 by the phenol red method. The concentration of SOS and Gpx was made by spectrometry. The analysis of TBARS by the Elisa method.

RESULTS

Results showed that the LBI was able to almost completely close the risk of the wound, similar to the control group. C2C12 cells were able to produce both NO and H2O2 one hour after the addition of the venom being statistically different from the control. LBI irradiation significantly reduced NO release, but did not alter the release of H2O2. No significant difference was observed of anti‐oxidant enzymes in C2C12 cells incubated to BjV. LBI Irradiation induced a significant increase of SOD in comparison to the venom group, but not modify Gpx release. BjV did not cause lipid peroxidation and LBI treatment did not modify this effect.

CONCLUSION

Photobiomodulation caused muscle cell protection and this protection seems to be related to decrease of reactive species of oxygen and nitrogen and increase of anti‐oxidants.

Support or Funding Information

Comissão de Aperfeiçoamento de Pessoal do Nível Superior (CAPES)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Sirt3 in Lipid Generation
Authors: Le Truc P , Ma Oanh , Talbott George , Ho Linh
Published: June 2021

Sirt3 is a major mitochondrial deacetylase which has important roles in regulation of metabolic homeostasis and metabolic diseases. ST2 cells are mesenchymal stem cells taken from bone marrow. The purpose of this research is to identify if Sirt3 plays a role in marrow adipogenesis to regulate insulin sensitivity through adipokines, especially adiponectin using ST2 cell models.

Particularly, we used the gain‐function of Sirt3 in ST2 cells to assess the role of Sirt3 in adipogenesis. We were able to successfully overexpress Sirt3 and differentiate ST2 cells into adipocytes. We found that the induction of Sirt3 leads to increased adipogenesis compared to controls. The increase in adipogenesis was in line with elevated triglyceride levels in Sirt3 overexpression compared to controls (P < 0.01 in day 5 and P < 0.05 in day 7 after differentiation). This data is consistent with the increase in percentage area of adipocytes, which was highly significant in Sirt3 overexpression than that in the controls using Oil Red O staining (P< 0.01). The induction of adipogenesis by Sirt3 is associated with increased gene expression of adipocyte markers as well as adiponectin/adipokines.

In addition, we inhibit Sirt3 function in ST2 cells using an inhibitor of Sirt3 called 3‐TYP. We treated cells with 50μM and 100M of 3‐TYP, versus control. Our results show that ST2 cells treated with Sirt3 inhibitor decreased adipogenesis by reduced area of adipocytes compared to controls especially at 100μM of 3‐TYP (P<0.05). Triglyceride measurements of 3‐TYP treated cells versus controls showed consistent results with reduced adipogenesis, especially at 100 μM on day 5 (p‐value <0.001). The expression levels of adipocyte markers and adiponectin/adipokines were statistically significantly reduced compared to the controls.

These statistically significant consistent data suggest that Sirt3 gene plays an important role in modulating adipogenesis and adiponectin/adipokines expression. We hope our study contributes to the efforts of revealing Sirt3 functions in metabolic homeostasis and diseases. The ultimate goal is to use Sirt3 as a potential target in treating insulin resistance and other metabolic abnormalities.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fascia of the Head & Neck and Spread of Infection
Authors: Grogan David , Spears Robert
Published: June 2021

While fascia can be found throughout the body, it plays a particularly important role in the head and neck. Although it serves as boundaries that separate compartments from one another, fascia also creates spaces that allow for the potential spread of infection from one location to another. The objective of this presentation will be to give a general understanding of the different fascial layers that exist within the head and neck, the structures they invest, and the spaces created by these layers. The fasciae of the neck consists of the investing layer of deep cervical fascia, prevertebral fascia, pretracheal fascia, and two carotid sheaths, which typically divide the neck into a musculoskeletal compartment, a visceral compartment, and two neurovascular compartments. The cervical fasciae will continue onto the head where it will surround structures of the superficial face, deep face, and head, creating numerous spaces with communication not only to themselves but also to the spaces of the neck. Thus, particular focus will be given on how infection can spread between the fascial planes and spaces of the head and neck and the inherent clinical importance of understanding the fasciae in this area.
VEGF mRNA levels are upregulated by lipopolyssacharide in the cervix of pregnant mice
Authors: Newell Allison , Thompson Morgan , Mowa Chishimba Nathan
Published: June 2021

Cervical remodeling (CR) is one of the few biological processes that entails physiological inflammation, and can be significantly enhanced by inflammation‐inducing agents, such as lipopolyssacharide (LPS), a component of bacteria cell wall. Infection is one of the leading causes of preterm labor, and CR and inflammation are both closely associated with microvascular alterations. Of note, we recently identified and characterized expression of a potent regulator of the vasculature, vascular endothelial growth factor (VEGF) and VEGF signaling molecules in the cervix of pregnant and parturient rats. Our long term goal is to investigate whether the mechanism(s) underlying LPS‐enhanced CR involve VEGF and alters microvasculature. Here, we sought to characterize the pattern of VEGF mRNA expression in LPS‐treated pregnant mice using PCR. LPS dissolved in normal saline was administered intra‐cervically in a dose‐dependent manner to three groups of mice at day 12 of pregnancy (n=3 per group), namely vehicle only, 250ng and 300ng. The cervix was harvested 6 hrs later and levels of VEGF mRNA were analyzed by PCR. Our preliminary data revealed that LPS up regulated levels of VEGF mRNA in a dose‐dependent manner. These studies show that LPS‐induced preterm labor involves enhanced VEGF synthesis, which may lead to abnormal microvascular remodeling and mobilization and infiltration of vascular‐derived CR mediators.
Functional analysis of p53 response elements in the human Polycystic Kidney Disease Gene 1 (PKD‐1) promoter
Authors: van Bodegom Diederik , Roessingh Wijnand , El‐Dahr Samir
Published: June 2021

We have previously shown that p53 represses human PKD1 expression in vivo and in vitro, and that this effect is dependent on histone deacetylase activity (JBC, 281, p.31234, 2006). The 5′‐regulatory region of PKD1 contains 4 putative p53 response elements (p53RE4‐1) at positions: −2.7kb, −1.2kb, −0.8kb and −0.2kb respectively. In this study, we delineate the differential roles of the p53RE's in transcriptional repression of the PKD1 gene. We performed site‐directed mutagenesis of distal p53RE4, proximal p53RE1. The wild‐type and mutant PKD1 promoter‐reporter constructs were co‐transfected with a p53 expression plasmid in IMCD3, HCT116(p53+/+) and HCT116(p53−/−) cells. p53RE4 deletion decreased PKD1 baseline promoter activity (2.4, 1.8 and 1.4‐fold) in IMCD3, HCT116(p53+/+) and HCT116(p53−/−) cells, respectively, and significantly enhanced p53‐mediated repression in all cell lines, indicating that p53RE4 is a positive cis‐response element. In contrast, deletion of the p53RE1 induced a 1.6‐fold increase in baseline PKD1 promoter activity; this was observed in p53+/+ but not p53−/ − cells. p53RE1 failed to mediate repression of a known p53‐target gene ‐the bradykinin B2 receptor‐ indicating that p53RE1 functions in a context‐specific manner. Chromatin Immunoprecipitation assays in IMCD3 cells revealed the DNA regions surrounding p53RE1/2 are occupied by HDAC 1 and 2. We conclude that the balance between p53RE2 and p53RE1 occupancy by p53 and HDACs determines the final response of PKD1 to p53 within a given cell.

Supported by NIH grant DK‐062250
Determination of affinity constants for binding of Salmonella flagellar export proteins FliH and FliI
Authors: Nguyen Viet Q. , Kraft Lewis J. , McMurry Jonathan L.
Published: June 2021

Interaction of Salmonella enterica FliH with FliI is critically important to flagellar assembly, a Type III secretion process. FliH is a negative regulator of FliI, the export ATPase. The two have been shown qualitatively to stably associate. To quantitatively analyze the interaction, a saturation binding study was undertaken using an ELISA‐based approach after repurifying complexes of purified His‐tagged FliH equilibrated with varying amounts impure FLAG‐tagged FliI overproduced in a cell lysate. KD was determined to be 119 +/− 14 nM. No binding was detected with an N‐terminal FliH truncation (residues 1–102). Binding was also assayed using a C‐terminal FliH truncation (residues 99–235) shown to bind less stably than full‐length FliH in copurification assays. Results support the veracity of the assay. The present work shows that the FliH‐FliI association is comparable in affinity to that between FliH and the flagellar structural protein FliN and will serve as a basis for analyzing more complex protein assemblies such as FliN‐FliH‐FliI, illuminating the dynamics of flagellar export. This work was supported by grants from the NIH (R15GM080701), the Research Corporation (Cottrell College Science Award) and the KSU Mentor‐Protégé Program.
Esculetin as a probe and substrate of bovine phenol sulfotransferase (bSULT1A1)
Authors: Beckmann Joe D. , DeGood Kate , Cassidy Patrick , Bauer Adam
Published: June 2021

Phenols are often conjugated by sulfotransferases (SULTs), with 3′‐phosphoadenosine−5′‐phosphosulfate (PAPS) as the donor substrate. Fluorescence energy transfer revealed a ternary complex of homodimeric bSULT1A1, PAP and 7‐hydroxycoumarin; however, unbound ligand signal confounded spectral interpretation. Therefore, we evaluated other (di)hydroxycoumarins as probes and substrates for recombinant bSULT1A1. 6,7‐Dihydroxycoumarin (DHC, esculetin) forms a highly fluorescent (λmax = 450 nm) PAP‐dependent complex with the enzyme. Titrations of DHC into SULT:PAP indicated half‐sites saturation with Kd = 0.22 μM. Inclusion of borate, to model the sulfuryl transfer transition, resulted in full‐sites binding with Kd = 1.7 μM. Reaction progress curves of DHC sulfation with excess PAPS were biphasic, emission spectra indicated two products, and initial rates indicated Km = 0.08 μM and kcat = 2.9 min−1. Rapid kinetics of DHC binding is fit by two first‐order rates: [DHC]‐dependent (200 sec−1), and a slower [DHC]‐independent phase (25 sec−1). This may result from initial binding of DHC followed by a protein conformation change. Esculetin is thus providing new information regarding SULT catalysis and protein dynamics.
Studies with S‐alkylated cysteine methyl esters suggest carboxylesterase 1 may be polyisoprenylated methylated protein methyl esterase
Authors: Lamango Nazarius Saah , Koikov Leonid N , Duverna Randolph
Published: June 2021

An estimated 2% of eukaryotic proteins are S‐polyisoprenylated on a terminal cysteine. The terminal ‐COOH is esterified by isoprenyl carboxyl methyl transferase (ICMT). The methyl esters are hydrolyzed by serine esterases in the only reversible step of the pathway. While ICMT has been well studied, the esterases have received little attention. We purified and identified a liver esterase, which like ICMT has an ER‐retention signal. This “promiscuous” esterase, so‐called because of its large and flexible active site and repertoire of substrates, is better known for its (pro)drugs and xenobiotics metabolism. If its endogenous substrates are polyisoprenylated proteins (PP) as suggested by earlier preliminary studies with farnesylated substrates, it will show a higher affinity for S‐farnesylated (C15) and S‐geranylgeranylated (C20). A series of chromophoric S‐alkylated substrates ranging from C2 to C‐20 were synthesized. Enzyme kinetics revealed an increase in affinity (Km values) for ethyl (505±63 μM), prenyl (294±25 μM), geranyl (87±12 μM), t,t‐farnesyl (29±2.2 μM) and all t‐geranylgeranyl (15±2.7 μM). Since the C15 and C20 modifications occur in proteins, the results with the latter two substrates suggest that PP may constitute the endogenous substrates of this esterase, with the “promiscuous” features designed to accommodate the polypeptides bearing the polyisoprenylated methylated cysteine.
C‐Terminal Residues of Glutathione Synthetase
Authors: Brown Teresa R. , Barelier S. , Cundari T. R. , Dinescu A. , Anderson M. E.
Published: June 2021

Glutathione (GSH:γ‐L‐Glutamyl‐L‐cysteinylglycine) is an important antioxidant found in living cells. Low cellular levels of GSH are associated with diseases such as stroke, AIDS and diabetes. Glutathione Synthetase (GS) catalyses the final ATP‐dependent step to form GSH. Human GS is the only mammalian member of the ATP‐grasp super‐family (an important class of amine carboxylate ligases) whose function is known; other family members are involved in nucleotide and cell wall synthesis. Each monomer of the dimeric GS has an active site, and GS displays negative cooperativity. There are three highly conserved loop regions (G‐,A‐,S‐loops). Higher eukaryotes have high A‐loop (alanine‐rich) identity (∼90%), while there is less identity in lower eukaryotes and prokaryotes (∼45%). The thirteen residue (Ile454‐Ala466) A‐loop is near the C‐terminal portion of GS and the glycine binding site. Several residues of the A‐loop were computationally modeled, mutated by site‐directed mutagenesis, and the mutant GS enzymes purified and assayed to characterize their role in GS function. We studied several GS mutants and found Glu 445 Arg and His 456 Ala enzymes have lowered activity (∼30% and ∼50%, respectively) compared to wild type GS. This A‐loop is important for GS activity. (Supported by a Welch Foundation Grant (Chem. Dept. TWU), U.S. Dept. Ed (CASCaM UNT, T.R.C.), Faculty Research Grant UNT (TRC).
Tracing Complex Neural Crest Cell Behaviors in the Embryo using Photoactivation of KikGR
Authors: Kulesa Paul , Teddy Jessica , Stark Danny , Smith Sarah , McLennan Rebecca
Published: June 2021

Key to our understanding of how molecular mechanisms modulate cell migratory patterns in the embryo is information about cell behaviors at precise times and spatial locations. However, it has been challenging to selectively mark subgroups of migratory cells in vivo without disruption to the local microenvironment. We apply a combination of photoactivation cell labeling using KikGR, and confocal time‐lapse imaging to investigate cell behaviors within the migratory front and trailing subgroups of chick neural crest (NC), a highly invasive pluripotent embryonic cell population. KikGR is a fluorescent protein that is electroporated into NC cells and contains a green chromophore that is photoconvertible to red following 405 nm laser excitation, thus allowing less invasive optical precision to fluorescently mark single and small groups of cells. We find that the spatial order of cranial NC cell emergence translates into a distal‐to‐proximal invasion of the target branchial arches. Lead and trailing NCCs display similar average cell speeds and directionalities. Surprisingly, we find that lead NCCs proliferate along the migratory route and grow to outnumber trailing NCCs by nearly 3 to 1. Our results suggest a model in which lead and trailing NCC behaviors are distinct and coordinated to maintain a spatially‐ordered invasion of the branchial arches. Research support is from the Stowers Institute.
Changing the Substrate Specificity of Creatine Kinase from Creatine to Glycocyamine: Evidence for a Highly Evolved Active Site
Authors: Barth Emily J , Fraga Dean , Edmiston Paul L
Published: June 2021

Eight variants of creatine kinase were created to switch the substrate specificity from creatine to glycocyamine using a rational design approach. Changes to creatine kinase involved altering several residues on the flexible loops that fold over the bound substrates including a chimeric replacement of the guanidino specificity loop from glycocyamine kinase into creatine kinase. A maximal 2,000‐fold change in substrate specificity was obtained as measured by a ratio of enzymatic efficiency (kcat/KM · Kd) for creatine vs. glycocyamine. In all cases, a change in specificity was accompanied by a large drop in enzymatic efficiency. This data, combined with evidence from other studies, indicate that substrate specificity in the phosphagen kinase family is obtained by precise alignment of substrates in the active site to maximize kcat/KM · Kd) as opposed to selective molecular recognition of one guanidino substrate over another. A model for the evolution of the dimeric forms of phosphagen kinases is proposed in which these enzymes radiated from a common ancestor that may have possessed a level of catalytic promiscuity. As mutational events occurred leading to greater degrees of substrate specificity, the dimeric phosphagen kinases became evolutionary separated such that the substrate specificity could not be interchanged by a small number of mutations.
Regulation of heme degradation in association with multiple sclerosis
Authors: Kralik Lubomir , Krasulova Eva , Hrdinka Matous , Pospisilova Lenka , Tyblova Michaela , Havrdova Eva , Martasek Pavel
Published: June 2021

Heme oxygenase‐1 (HO‐1) is a stress protein that degrades heme to biliverdin, iron, and carbon monoxide (CO). A (GT)n dinucleotide repeat polymorphism in the HO‐1 promoter has been shown to modulate HO‐1 gene expression. Short (<25) GT repeats are associated with HO‐1 up‐regulation. Multiple sclerosis (MS) is an autoimmune disorder resulting in CNS demyelination and neurodegeneration. HO‐1 or exposure to its end product CO counters autoimmune neuroinflammation and might have therapeutic potential for the treatment of MS. In this study, we investigated the association of the HO‐1 promoter (GT)n repeat polymorphism in 291 patients with MS. We used data from previous studies as a control group. We compared the frequencies of short (<25) repeat (class S) and long (≥25) repeat (class L) alleles. No significant difference between these two groups was found. Thus, the (GT)n dinucleotide repeat polymorphism in the HO‐1 promoter is not associated with MS. Comparison of the course of MS disease in patient groups homoallelic for either S/S or L/L may add yield differences in the expression of the diseases symptoms, suggesting that therapeutic intervention with HO‐1 inducers may be possible.

Supported by Grants: IGA MZ 1A8713‐5, GAUK 109/2006, MSMT 0021620806.
Effect of fucoidan on mRNA and promoter activity of matrix metalloproteinases(MMPs) in skin fibroblasts
Authors: Moon Hee Jung , Shim Sun ‐ Nyu , Ku Mi‐Jeong , Lee Yong‐Hwan
Published: June 2021

In the present study, to find molecular mechanism of UVB induced matrix metalloproteinase (MMPs) in skin fibroblasts, we investigated the effect of fucoidan on the MMP‐1 mRNA level and promoter activity. Fucoidan treatment of 0, 1, 10, 100 μg/mL concentration. The cells were incubated for 24 hr and after UVB irradiation, they were then further harvested. mRNA expression of MMPs was estimated by using RT‐PCR. The cells pretreated with fucoidan were inhibited in the expression of MMP‐1 at mRNA levels in the following order; 100 μg/mL> 10 μg/mL > 1 μg/mL compared to the control. In order to study the inhibitory effects of fucoidan on UVB‐induced MMP‐1 expression at transcription level, luciferase assay was performed using pGL3 MMP‐1 construct. To examine the roles of UVB in MMP‐1 promoter activity, a luciferase assay was performed at 0, 6hr, 12hr and 24hr after UVB (100 mJ/cm2) exposure. MMP‐1 promoter activity was maximally induced at 6hr after UVB irradiation and subsequently declined to basal levels in cells.

In conclusion, fucoidan may down‐regulated MMP‐1 promoter and induce inhibition of MMP‐1 mRNA. Through this mechanism, fucoidan may have anti‐photoaging effect.

*This work was supported by the grant of KICOS
Characterization, cloning and analysis of the promoter region of the SNAT2 gene of the rat.
Authors: Ortiz Victor , Aleman Gabriela , Escamilla Martin , Recillas‐Targa Felix , Torres Nimbe , Tovar Armando R
Published: June 2021

SNAT2 is a Na+‐dependent transporter of the SLC38 gene family. SNAT2 is expressed ubiquitously in mammalian tissues and transport neutral amino acids. However, little is known about the regulation of its expression. As an initial study of the molecular regulation that control its expression we cloned a 1.8 Kb of the SNAT2 5′ flanking region. Transcription initiation site was located at 970 pb from the translation start site by 5′RACE and primer extension. Analysis with the MatInspector software to identify putative transcription‐factor‐binding sites reported TATA box, CREB, AP‐1 and ATF consensus sites in this region. Constructs containing progressive 5′ deletions were ligated in a luciferase reporter vector for functional studies in HepG2 cells. Region −1792 to −336 showed 30 to 50 fold promoter activity over the empty vector. Promoter activity was greatly increased when transfection was performed with forskolin and PMA. Activation of SNAT2 gene is possibly controlled by CREB and ATF response elements located at −368 pb and −62 pb or maybe by a AP‐1 response element located at −532 pb. The results showed that the SNAT2 gene expression is regulated via PKA and PKC signal pathway. CONACyT 46134‐M.
Enzymes involved in the formation of 4‐hydroxy‐acyl‐CoA esters from γ‐hydroxybutyrate (GHB) and γ‐hydroxypentanoate (GHP)
Authors: Zhang Guofang , Kasumov Takhar , David France , Anderson Vernon E. , Brunengraber Henri
Published: June 2021

GHB is both a brain metabolite and a drug of abuse (date rape drug). GHP, a GHB analog, is also a drug of abuse. During EB 2007, we reported the identification of 4‐phospho‐butyryl‐CoA (P‐GHB‐CoA) and 4‐phospho‐pentanoyl‐CoA (P‐GHP‐CoA) in liver, kidney and brain of rats injected with GHB or GHP. We perfused livers with fatty acids (C3 to C5) hydroxylated on various carbons. HPLC‐MS‐MS‐ESI analysis showed that phosphoacyl‐CoAs are formed only from 4‐hydroxyacids. Using LC‐MS/MS assays, we searched for liver enzymes involved in the formation of P‐GHB‐CoA and P‐GHP‐CoA. Enzyme activity catalyzing the formation of GHB‐CoA or GHP‐CoA from the free acids is present in cytosol, mitochondria and microsomes. The phosphorylation of GHB‐CoA or GHP‐CoA occurs only in the cytosol, and is concentrated in the 15 to 35% ammonium sulfate fraction. Supported by NIH grant ES013925.
HIF‐1 is not a critical determinant for metabolic zonation in liver acinus
Authors: Goda Nobuhito , Ochiai Daigo , Fujiki Natsuko , Johnson Randall S. , Suematsu Makoto
Published: June 2021

In liver, hepatocytes show zonal expression of enzymes involved in carbohydrate metabolisms depending on their localizations in acinus. Among factors responsible for the metabolic zonation, oxygen has been thought to play a critical role for hepatocytes to express distinct enzymes by sensing changes in oxygen concentrations in liver acinus. This study aims to elucidate whether hypoxia inducible factor (HIF)‐1, an oxygen‐sensitive transcription factor, serves as a molecular determinant in the establishment of metabolic zonation of hepatocytes among liver acinus. To this end, we inactivated gene of HIF‐1α, a subunit of HIF‐1 that control its transcriptional activity, in a hepatocyte‐specific manner in mice. HIF‐1α (−/−) mice are viable without any apparent abnormalities of development. Immunohistochemical analysis revealed that expressions and distributions of carbohydrate‐related enzymes including glucokinase (GK) were comparable between control and HIF‐1α (−/−) mice. Exposure to 8 % hypoxia induced expressions of pyruvate kinase and GK greatly as a function of time in isolated control hepatocytes, while the induction was delayed and attenuated in HIF‐1α‐deficient hepatocytes. These results suggest that HIF‐1 is not essential for the maintenance of metabolic zonation in liver acinus, but is required for hypoxic responses of hepatocytes in enzyme expressions.
Conditional knock‐down of the transcriptional coactivator PGC‐1α in mice using Cre‐LoxP induced RNA interference
Authors: Bagattin Alessia , Hugendubler Lynne , Hayes Schantel , Mueller Elisabetta
Published: June 2021

PGC‐1α (peroxisome proliferator‐activated receptor gamma coactivator 1α) is a transcriptional coactivator involved in mitochondrial biogenesis and energy metabolism. The function of PGC‐1α has been studied in vivo through the generation of tissue‐specific transgenic and knock‐out models. We have used RNAi technology to generate transgenic mice with a conditional knock‐down of PGC‐1α in order to study the contribution of PGC‐1α in the response to various metabolic challenges. Mice carrying the U6‐ploxPneo‐PGC‐1α‐RNAi transgene were crossed with EIIa‐Cre transgenic mice to ubiquitously knock‐down PGC‐1α expression in vivo. U6‐PGC‐1α‐EIIa‐Cre mice were born at the expected Mendelian ratio. Upon cold exposure, PGC‐1α knock‐down transgenic mice exhibit reduced PGC‐1α mRNA induction in brown fat compared to controls and, as previously shown in knock‐out mice, they have decreased UCP1 (uncoupling protein 1) mRNA levels. Furthermore, microscopic analysis of H&E stained brown fat tissue sections revealed accumulation of large lipid droplets, as previously observed in PGC‐1α knock‐out animals. In conclusion, we have developed an animal model that mirrors the total PGC‐1α knock‐out mouse and that allows investigation of PGC‐1α function in vivo through a system that does not require the lengthy process of gene‐targeting nor ES cell manipulation.

Supported by the Intramural Research Program of NIDDK, NIH.
The phosphatase laforin crosses evolutionary boundaries and links carbohydrate metabolism to neuronal disease
Authors: Dixon Jack E , Gentry Matthew S , Dowen Robert H , Worby Carolyn A , Mattoo Seema , Ecker Joseph R
Published: June 2021

Protein Tyrosine Phosphatases (PTPases) are a large family of catalyst that play critical roles in regulating reversible phosphorylation. Mutations in specific PTPases can have devastating effects. Lafora disease (LD) is a progressive myoclonic epilepsy resulting in severe neurodegeneration followed by death. A hallmark of LD is the accumulation of insoluble polyglucosans called Lafora bodies (LBs). LD is caused by mutations in the gene encoding the phosphatase laforin, which reportedly exists solely in vertebrates. We utilized a bioinformatics screen to identify laforin orthologues in five protists. These protists evolved from a progenitor red alga and synthesize an insoluble carbohydrate whose composition closely resembles LBs. Furthermore, we show that the kingdom Plantae, which lacks laforin, possesses a protein with laforin‐like properties called starch excess 4 (SEX4). Mutations in the Arabidopsis thaliana SEX4 gene results in a starch excess phenotype reminiscent of LD. We demonstrate that Homo sapiens laforin complements the sex4 phenotype and propose that laforin and SEX4 are functional equivalents. Finally, we show that laforins and SEX4 dephosphorylate a complex carbohydrate and form the only family of phosphatases with this activity.
A DNA Combinatorial Library of 258 Strand Combinations for DNA Computing
Authors: Waters Colin , Macula Anthony , Pogozelski Wendy
Published: June 2021

DNA‐based computers use combinatorial libraries of DNA strands to store information while manipulating these strands to solve computationally hard problems in parallel. In order for DNA computing to be successful, these libraries must be constructed in an easy and reliable manner.

We show the construction of two libraries of 2exp3 and 2exp5 strand combinations and then we link them to produce a library of 2exp8 or 256 combinations of 150‐bp DNA strands. We then verify that the expected strands are present. This library should be useful in solving mathematical satisfiability (SAT) problems.
Ubiquitination of the polyglutamine disease protein ataxin‐3 enhances its activity as a deubiquitinating enzyme
Authors: Todi Sokol V , Winborn Brett J , Williams Aislinn J , Paulson Henry L
Published: June 2021

Protein ubiquitination regulates a broad spectrum of cellular functions. Diverse ubiquitin ligases and deubiquitinating enzymes (DUBs) control the ubiquitination state of, and types of ubiquitin chains attached to, various proteins. Ataxin‐3 (AT3) is a DUB with a polyglutamine tract which, when expanded, causes the neurodegenerative disorder Spinocerebellar Ataxia Type 3. Disease pathogenesis in expanded polyglutamine disorders is greatly influenced by the disease protein context and function, therefore understanding normal properties of AT3 is important. Previous data from our laboratory and others revealed that AT3 functions in protein quality control. Here we provide evidence that AT3 ubiquitination enhances its DUB activity. In cells, endogenous AT3 ubiquitination is increased by the presence of excess ubiquitin chains, suggesting that AT3 ubiquitination is linked to cellular ubiquitin levels. Ubiquitinated AT3 (AT3‐ub) cleaves lysine(K)‐63 hexaubiquitin chains more efficiently than does unmodified AT3. Similar to unmodified AT3, AT3‐ub preferentially cleaves K‐63 linked ubiquitin over K‐48 chains, and longer over shorter chains. Collectively our findings suggest that ubiquitination of AT3 and the corresponding enhancement of its DUB activity are important to cells in which protein homeostasis is impaired.

This work was supported by: NRSA NS056609 (AJW), R01 NS038712 (HLP).
Overview of drug discovery and development: traditional and evolving roles of academic institutions
Authors: Conn P. Jeffrey
Published: June 2021

Despite tremendous progress in furthering our understanding in biology, chemistry, and related disciplines there has been less progress in translation of these advances in basic science to discovery of new therapeutic agents. There is a critical need to focus increased attention on more fully validating potential new drug targets and allow basic science discoveries to be most useful in supporting full drug discovery efforts in industry settings. For the biomedical research enterprise to be maximally effective in translating new basic science discoveries into breakthrough new therapeutic agents, it will be important for scientists in academic and other non profit settings to increase efforts to critically evaluate novel hypotheses related to new drug targets and provide data needed to a allows translation of advances in basic science to full drug discovery programs. This short presentation will provide an introduction to the symposium by outlining the drug discovery and development process and suggesting approaches by which academic scientists may contribute to determining “drugability” and potential validation of potential new drug targets. The following presentations will provide examples of early stage drug discovery efforts being pursued by academic scientists.