bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA‐binding Triggers the Tetramerization of the Glucocorticoid Receptor in Live Cells
Authors: Presman Diego M. , Ganguly Sourav , Schiltz R. Louis , Karpova Tatiana , Hager Gordon L.
Published: November 2023

Transcription factors dynamically bind to chromatin and are essential for the regulation of genes. While a large percentage of these proteins appear to self‐associate to form dimers or higher‐order oligomers, the stoichiometry of DNA‐bound transcription factors has been poorly characterized in vivo. The glucocorticoid receptor (GR) is a ligand‐regulated transcription factor widely believed to act as a dimer. However, this paradigm has been established exclusively from in vitro studies. Using fluorescent imaging techniques and a cell line containing a tandem‐array of steroid response elements, we analyzed the oligomerization state of the GR in vivo. We show that while GR is mostly dimeric in the nucleoplasm, it presents higher oligomerization states, predominantly tetramers, when bound to the array. Consistent with these findings, a point mutation that mimics DNA‐induced conformational changes promotes higher oligomerization in the entire nucleoplasm. We therefore propose that DNA allosterically modulates the oligomerization state of GR, triggering a novel quaternary structure (i.e. a tetramer), which would be the truly active form of the receptor. Since GR's oligomerization state has been implicated in therapy outcome, our findings open new doors to the rational design of novel GR ligands.

Support or Funding Information

This research was supported by grants from the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.
Bioinformatics and Electrophoretic Mobility Shift Assays to Characterize Activators and Repressors of the Lupinus albus Formamidase Gene
Authors: Amadi Michael Chigoziem , Galang Nino Pierre , Sin David , Uhde‐Stone Claudia
Published: November 2023

Background

Lupinus albus (white lupin) is a legume adapted to grow under low phosphorous and nitrogen conditions. Phosphorous and nitrogen are important components of cellular molecules such as nucleic acids, amino acids, membrane phospholipids, and ATP. Reserves of the non‐renewable element phosphorus are on a steep decline due to over mining. Nitrogen is the most limiting nutrient of much of the world's agricultural land. We previously identified white lupin genes that showed induced expression under both phosphorus and nitrogen deficiency. Among these genes, a formamidase‐encoding gene in white lupin, named LaFmd, was induced under both nutrient deficiencies. Using promoter deletion analysis, we previously identified putative activator and inhibitor‐binding regions within the formamidase (LaFmd) promoter.

Method

White lupin was grown hydroponically under phosphate deficiency, nitrogen deficiency, and as nutrient‐sufficient control. Protein was isolated from roots, using the P‐Per plant protein isolation kit (Thermo Fisher, Waltham, MA). Computational analysis (PlantPan 2.0 and PlantCARE) was performed to identify known regulatory DNA motifs. Biotin‐labeled DNA oligonucleotides of the hypothesized regulatory regions were ordered (ELIM Biopharm, Hayward, CA) to perform Electrophoretic Mobility Shift Assays.

Results

Bioinformatics analysis identified putative regulatory elements within the hypothesized activator and inhibitor‐binding regions of the LaFmd promoter, including TFmatrixID_0051 and TFmatrixID_0130. Oligonucleotides spanning these regulatory elements were designed and ordered, and will be used for Electrophoretic Mobility Shift Assays. A band shift with protein from nutrient‐deficient conditions, but not nutrient‐sufficient control, would indicate binding of an activator. A band shift only under nutrient sufficiency would indicate binding of a repressor.

Conclusion

The outlined approach will be used to identify transcription factors involved in regulating the formamidase gene under phosphorus and nitrogen deficiency.
A Genome Editing Approach to Studying Pmp22 Enhancer Functionality
Authors: Pantera Harrison , Moran John , Lopez‐Anido Camila , Svaren John
Published: November 2023

The function of the peripheral nervous system is dependent on myelination of nerves by Schwann cells. This process is tightly governed by a network of transcription factors during development. Myelin stability is adversely affected in the most common form of the hereditary peripheral neuropathy called Charcot‐Marie‐Tooth Disease. This form, classified as CMT1a, is caused by a 1.4 Mb duplication on chromosome 17. This duplication includes the abundantly expressed myelin gene Peripheral Myelin Protein 22 (Pmp22), which is associated with multiple disease states in a gene dosage‐dependent manner. Previous studies have shown that reducing the expression of Pmp22 in rodent models of CMT1A results in amelioration of the neuropathy phenotype found in myelinating Schwann cells in the PNS. In recent work characterizing the mechanisms regulating Pmp22 transcription, we identified a cluster of enhancers approximately 100 kb upstream of the Pmp22 TSS. This upstream cluster was found within a smaller duplication identified in patients with CMT1A‐like symptoms, where the Pmp22 coding region itself was not part of the duplication. This cluster possesses multiple binding sites for factors that are required for Schwann cell development and myelination: Egr2 and Sox10. Previous studies in our lab implicate these sites as Pmp22 enhancers, but until now there has been an absence of evidence of functional importance in achieving the high endogenous levels of Pmp22 expression. Using genome editing tools including CRISPR/Cas9 in a modified rat Schwann cell line that expresses Pmp22 at near‐physiological levels, we have established clonal lines possessing deletions that include these upstream transcription factor‐binding sites. Preliminary data show an observed decrease in Pmp22 transcript expression ostensibly due to the loss of these sites. These data show for the first time the requirement of these upstream enhancers for full Pmp22 expression and alternate promoter usage.

Support or Funding Information

This work is supported by NIH grant R01 NS083841.
Maternal Versus Lifelong High‐Fat Diet Differentially Alters Genome‐Wide DNA Methylation in Rat Liver
Authors: Moody Laura , Kriska Adam , Chen Hong , Pan Yuan‐Xiang
Published: November 2023

Early life diet programs offspring such that discrepancy between intrauterine and postnatal nutrition will have negative consequences later in life. Specifically, exposure to high‐fat diet (HF) at critical developmental stages is potentially associated with metabolic syndromes; however, it is unclear which epigenetic mechanisms are responsible for such outcomes. We hypothesize that due to differences in DNA methylation, a mismatched perinatal diet and post‐weaning diet is more metabolically detrimental than a lifelong HF diet. In the present experiment, male Sprague‐Dawley rats were exposed to HF diet during gestation and lactation. At weaning, half the animals were kept on the HF diet (HF/HF) and half were switched to a control AIN‐93G diet (HF/C). Complementary Methylated DNA Immunoprecipitation coupled with high‐throughput sequencing (MeDIP‐seq) and Methylation‐sensitive Restriction Enzyme digestion followed by sequencing (MRE‐seq) were used to quantify DNA methylation in the liver. We identified 3,966 differentially methylated regions (DMRs) between HF/C and HF/HF groups. Of these DMRs, 35% were mapped to gene bodies while 6% fell within promoter or downstream regions. Of the differentially methylated genes, 21 were clustered in the type II diabetes mellitus and the adipocytokine signaling pathways, two metabolically relevant systems that are known to respond to HF intake and lead to insulin resistance. Quantitative PCR showed altered expression of many genes in these pathways, including carnitine palmitoyltransferase 1A (Cpt1a), tumor necrosis factor receptor (Tnfrsf1b), retinoid X receptor (Rxra and Rxrb), and acetyl‐CoA carboxylase (Acacb). The remaining 59% of DMRs were not associated with protein coding genes. Sixteen of these DMRs were mapped to non‐coding RNAs including 9 miRNAs, 9 snRNAs, 6 snoRNAs, 1 telomerase, and 1 RNase P. Interestingly, several genes in the type II diabetes mellitus and the adipocytokine signaling pathways were predicted targets of these differentially methylated miRNAs. Our results indicate that compared to a lifelong HF diet, exposure to mismatched early and late life diets induces DNA methylation that impairs insulin‐ and lipid‐related pathways. Furthermore, methylation and expression of miRNA may provide an additional level of epigenetic regulation that drives these metabolic outcomes.

Support or Funding Information

UIUC Research Board grant #12192; Data Purchase Program from University of Illinois Library
Steroid Receptors can Activate Unique FoxA1 Binding Patterns through a Highly Dynamic Mechanism Associated with a Fast DNA Residence Time
Authors: Swinstead Erin E , Miranda Tina B , Paakinaho Ville , Baek Soongjoon , Hawkins Mary , Karpova Tatiana , Ball David , Mazza Davide , Lavis Luke D , Morisaki Tatsuya , Grøntved Lars , Presman Diego M , Hager Gordon L
Published: November 2023

The transcription factor (TF) FoxA1 has been implicated in steroid receptor (SR) binding patterns serving as a pioneer factor; however, the effects of SRs on the function of FoxA1 have been controversial. Classically it has been proposed that FoxA1 has a slow residence time on DNA allowing chromatin to remain open permitting access of SRs to binding sites. Conversely, it has recently been identified that SRs can alter the binding landscape of each other facilitating selective access to the chromatin by a mechanism termed dynamic assisted loading. To determine if this phenomenon extends to FoxA1 we characterised the estrogen receptor (ER), glucocorticoid receptor (GR), and FoxA1 in three breast cancer cell lines. Genome‐wide analysis of ChIP and DNaseI hypersensitivity assays upon hormone treatment show that both SRs can recruit FoxA1 to specific binding sites associated with an increase in chromatin accessibility. This indicates chromatin reorganization upon activation of ER and GR and recruitment of FoxA1 binding through an assisted loading mechanism. Most importantly, we have established the DNA residence time of FoxA1 by single‐molecule tracking in living cell at a rate of ~8–10 seconds. This finding is supported by the lack of a detectable FoxA1 DNase footprint together indicating fast chromatin interactions. These results suggest that many TFs in a given cell have the potential to affect the binding landscape of other TFs, depending on the chromatin context. In addition, this study has shifted our classical understanding of pioneer factors in breast cancer, demonstrating that steroid receptors can alter the response of FoxA1 though highly dynamic and fast DNA interactions.

Support or Funding Information

This research was supported by grants from the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.
Interactions of a Fluorescently‐Labeled Tobacco Mosaic Virus (TMV) RNA with Pokeweed Antiviral Protein (PAP)
Authors: Rodriguez David J , Domashevskiy Artem V
Published: November 2023

The 5′‐terminal 7‐methylguanosine (m7GpppN, where N is any nucleotide) cap structure of eukaryotic mRNAs is involved in RNA stabilization, transport, translation, and is recognized by many cellular proteins involved in pre‐mRNA splicing, RNA export, translation initiation and RNA turnover. Enzymatic production of capped RNA is an easy way to improve the stability and translational competence of RNA used for in vitro studies. Fluorescence spectroscopy is widely used to study these protein‐RNA interactions. The presence of a fluorescent cap derivative incorporated into the 5′ end of RNA provides an alternative method to study these cap‐binding interactions, and has several advantages: in general, fluorescent derivatives have a greater intensity than intrinsic protein (and/or nucleic acid) fluorescence; absorbance and emission maxima of the extrinsic fluorophore are far removed from native protein fluorescence, allowing interpretation of the interactions involving several proteins. Here, we describe a novel synthesis of a fluorescent cap analog, Anthranioyl‐GTP (Ant‐GTP), an in vitro cap labeling of a transcribed tobacco mosaic virus (TMV) RNA catalyzed by the recombinant vaccinia virus capping enzyme to produce functional Ant‐m7GpppG‐capped RNA. We further demonstrate efficient translation of this fluorescent RNA and study its interactions with pokeweed antiviral protein (PAP). PAP is a plant ribosome inactivating protein (RIP) produced by the common pokeweed plant. RIPs are RNA N‐glycosidases, and enzymatically remove purines from a universally conserved sarcin/ricin loop.
Characterization of the RNA‐binding Motif of Phosphoglycerate Kinase 1 – a Moonlighting Function for a Metabolic Enzyme
Authors: Walker Austin , Friend Kyle
Published: November 2023

Phosphoglycerate Kinase 1 (PGK1) is an enzyme that plays an integral role in glucose metabolism, catalyzing the reaction that converts 1,3‐bisphosphoglycerate into 3‐phosphoglycerate in glycolysis, and the reverse reaction in gluconeogenesis. In addition, PGK1 has been shown to have an RNA binding activity. This suggests that, in addition to its role in metabolism, PGK1 “moonlights” as a RNA‐regulatory enzyme. In particular, PGK1 was shown to down‐regulate the expression of the mRNAs which it bound. The goal of the current study is to identify the RNA sequence(s) that binds PGK1, using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. By identifying PGK1's consensus sequence, we will further elucidate the role of the enzyme in the cell beyond its metabolic capabilities, perhaps uncovering a new layer of regulation. We will then test how well PGK1 binds these RNA sequences via fluorescence polarization.

Support or Funding Information

Dr. G. Ashley Allen ‘65 Students Research Grant Fund, Washington and Lee University Chemistry and Biochemistry Department, Lenfest Grant
An Investigation of the Role of the NuA4 and Swr1 Complexes in Coordination of Transcription and RNA Splicing
Authors: Avitto Alexa N , Paripati Nikita , Moazamian Aron , Martinez Jordan , Moazamian Ryan , Pleiss Jeffrey , Kress Tracy
Published: November 2023

RNA splicing is a critical step in gene expression in which non‐protein coding introns are removed from newly transcribed RNA. RNA splicing is carried out by the dynamic spliceosome and may occur directly after or concurrent with transcription, suggesting a close relationship between these processes. Indeed, there is mounting evidence that transcription and RNA splicing are coordinated, however the mechanistic details underlying this coordination are still lacking. The NuA4 protein complex is a histone acetylation transferase (HAT) with known functions in transcription. NuA4 works in conjunction with Swr1, an ATP‐dependent chromatin remodeling enzyme. Histone acetylation by NuA4 recruits Swr1, which subsequently exchanges H2A for the variant histone H2AZ to activate transcription. Together, these complexes regulate the transcription of ribosomal protein genes (RPGs), which comprise a substantial proportion of the genes that contain introns in Saccharomyces cerevisiae. Thus, NuA4 and Swr1 are perfectly positioned to link transcription and RNA splicing. We identified genetic interactions between genes encoding spliceosome proteins and genes encoding components of the NuA4 (eaf3Δ, eaf7Δ) and Swr1 complexes (vps72Δ and swr1Δ), as well as HTZ1, which encodes H2AZ. Splicing‐specific microarray analysis and reverse transcription‐PCR studies revealed splicing defects in swr1Δ, eaf7Δ, and htz1Δ. In addition, as predicted by our genetic studies, the splicing defects in strains harboring deletions in splicing proteins were exacerbated by deletion of EAF7, SWR1 or HTZ1. These data support a role for NuA4 and Swr1 in RNA splicing. We are currently using chromatin immunoprecipitation assays to test whether NuA4 and Swr1 increase splicing efficiency by recruiting splicing proteins during transcription. Our preliminary results indicate that splicing factor recruitment is altered in the absence of EAF7. Together, these data support a model in which the NuA4 and Swr1 chromatin modification complexes interact with the splicing machinery to coordinate transcription and splicing.

Support or Funding Information

Cottrell College Science Award from the Research Corporation for Science Advancement (#20186)
Histone H3K36 Methylation is Required for Efficient Pre‐mRNA Splicing in Saccharomyces cerevisiae
Authors: Flood Danielle M , Kennedy Margaret C , Sorenson Matthew , Ucles Stefanie , Jha Deepak K , Strahl Brian D , Stevens Scott W , Kress Tracy L
Published: November 2023

Proper gene expression involves multiple steps, including RNA splicing where non‐protein coding regions of RNA are removed from the RNA transcript. RNA splicing is carried out by the large and dynamic spliceosome, which is comprised of small nucleoprotein complexes (snRNPs) that assemble on an RNA molecule in a precise order. Assembly of the spliceosome occurs co‐transcriptionally and RNA splicing is tightly coordinated with transcription to ensure precise and efficient gene expression. However, the mechanisms that underlie this coordination are poorly understood. In order to identify proteins that function to coordinate RNA splicing with transcription we carried out a genetic interaction study using Saccharomyces cerevisiae. We identified negative genetic interactions between genes encoding RNA splicing factors and the SET2 gene, a histone methyltransferase that methylates lysine36 on histone H3 (H3K36) to regulate transcription. Furthermore, we show that mutations that block H3K36 methylation also have negative genetic interactions with splicing factor genes, suggesting that H3K36 methylation is important for RNA splicing. Indeed, we have shown that deletion of SET2 or point mutation of H3K36 inhibits RNA splicing and exacerbates splicing defects in yeast strains harboring deletions of splicing factor genes. Using chromatin immunoprecipitation, we demonstrate that deletion of SET2 reduces the association of snRNPs with chromatin. Furthermore, we tested for reciprocal/reverse coordination between RNA splicing and transcription. We found that mutation of Prp28, a helicase protein that normally promotes RNA splicing, alters RNA polymerase II association with DNA. We are currently testing whether perturbation of RNA splicing alters H3K36 methylation. Thus, we provide evidence that H3K36 methylation is required for appropriate RNA splicing in yeast and suggest a model in which Set2 or H3K36 methylation help to recruit splicing factors to RNA during transcription.

Support or Funding Information

Cottrell College Science Award from the Research Corporation for Science Advancement (#20186) to T.L.K

American Cancer Society (RSG‐05‐137‐01‐GMC) to S.W.S National Institutes of Health (GM084246 to S.W.S and GM110058 to B.D.S) Cancer Prevention & Research Institute of Texas (RP101501) to M.R.S

Gateway to Graduate School funded by The National Science Foundation (NSF‐URM) to D.M.F
Genetic and Biochemical Studies on the Role of Essential Protein Dib1 in Pre‐messenger RNA splicing
Authors: Bowman Emily , Hernandez Cody , Schreib Christian , Lucas Amber , Maeder Corina
Published: November 2023

Pre‐messenger RNA splicing is a process that involves the removal of non‐coding regions of RNA and the ligation of protein‐coding regions in order to become a mature messenger RNA. This process involves the sequential addition of small nuclear ribonucleic protein particles, or snRNPs, that come together to form a pre‐catalytic spliceosome. Once formed, the U1 and U4 snRNPs leave to create the catalytically active spliceosome. The timing and regulation of this step is critical for splicing which must be accurate to a single nucleotide precision in order to prevent drastic effects further downstream in the translation of mRNA into protein.

A key figure in this process is Dib1, a small 17kDa essential protein. Dib1 has been shown to be necessary for cell viability and critical for splicing, although its role in splicing has yet to be characterized. Based on the recent cryo‐EM map of the U4/U6‐U5 triple snRNP complex, Dib1 appears to occupy a space in close proximity of the loop region of the U5 snRNA. This snRNA is expected to interact with the pre‐mRNA in the catalytically active spliceosome. Thus, in order for the spliceosome to be activated, Dib1 would need to depart from its location. To examine the role of Dib1 in splicing we have created multiple alleles of the dib1 gene in order to study their effects on cell viability in S. cerevisae. Using these strains, we are exploring mutant growth phenotypes and the effects on in vitro splicing. In addition, we are examining the effects of the mutations on the protein's stability.

Currently we have identified two temperature sensitive mutants through our mutational analysis. Using in vitro splicing assays, both were found to have splicing defects, although their defects differed. In order to determine why the splicing phenotypes differed, we are characterizing the effects of the mutations on the structure of Dib1. The proteins were purified using affinity chromatography followed by anion exchange chromatography. CD analysis of the purified protein was then performed. We will present these findings and discuss the implications on pre‐mRNA splicing.
Investigating the Mechanism by which the Small RNA MtlS Regulates the Mannitol Protein Transporter at the Post‐Translational Level in Vibrio cholerae
Authors: Hansen Elisabeth E , Coyle Maxwell C , Liu Jane M
Published: November 2023

Cholera, caused by the gram‐negative, halophilic bacterial pathogen Vibrio cholerae, remains a world health problem, causing millionsof deaths each year. Throughout its life‐cycle, V. cholerae is able to adapt to avariety of environments, from aquatic reservoirs to the human small intestine. This remarkable adaptability is likely due in part to gene regulation by small regulatory RNAs. We are examining one such regulatory pathway by studying the proteolysis of MtlA, the mannitol‐specific phosphotransferase system transporter. In the absence of environmental mannitol, the small RNA MtlS down‐regulates mtlA expression at the translational and post‐translational level. However, the mechanism by which MtlS post‐translationally directs MtlA proteolysis is currently unknown. Our lab has shown that MtlS induction significantly affects the expression of various proteins, which we hypothesize may be involved in MtlA proteolysis. Specifically, proteins up‐regulated by MtlS induction may act as proteases or protease adaptors. Proteins down‐regulated by MtlS induction may normally act to stabilize MtlA, such that when they are suppressed, MtlA degradation can occur. Currently, we are studying the potential role of three proteins—VC1899, GroL2, and VC1872—in MtlA proteolysis. VC1899 and GroL2 are both up‐regulated by MtlS induction, while VC1872 is down‐regulated. We created strains that overexpress or have knocked out one of each of these proteins, and analyzed the affect that these proteins have on MtlA levels over time in mannitol(MtlS‐repressing) and mannitol‐free (MtlS‐inducing) conditions. Additionally, we created knockout strains containing an arabinose‐inducible plasmid bearing MtlS, as an alternative method to analyze MtlA levels over time upon MtlS induction. Preliminary results suggest that VC1899, an uncharacterized protein thought to be involved in nucleic acid binding, represses MtlA protein levels. Single‐knockout mutants of either GroL2, an ortholog to the essential chaperone GroEL, or VC1872, a nun characterized protein with homology to serine protein kinases, do not display significant phenotypes with regard to cell growth or MtlA proteolysis. However, we are in the process of making double knockout mutant strains in order to test whether GroL2 and VC1872 may work in concert. Ultimately, gaining a better understanding of the mechanism of MtlA proteolysis could help to illuminate other regulatory systems in V. cholerae and other pathogens.

Support or Funding Information

NIH grant R15AI090606 to J.M.L.
Unraveling the Activation Mechanism of Cullin‐RING Ubiquitin Ligases using Quantitative Cross‐linking Mass Spectrometry
Authors: Yu Clinton , Mao Haibin , Novitsky Eric , Rychnovsky Scott D. , Zheng Ning , Huang Lan
Published: November 2023

Cullin‐RING ubiquitin ligases (CRLs) represent a superfamily of heterogeneous multi‐protein E3 ubiquitin ligases comprised of a cullin and RING protein catalytic core that mediate the ubiquitination of protein substrates. The full enzymatic activity of CRLs requires cullin modification by Nedd8 (i.e. neddylation). This ubiquitin‐like modification promotes both E2 recruitment and subsequent ubiquitin transfer, thereby stimulating the E3 activity of CRL ligases. Thus, neddylation represents a critical post‐translational modification that regulates protein ubiquitination by these complexes. Despite extensive structural studies on CRLs, certain structural details of CRLs during protein ubiquitination remain elusive. It has been shown that deamidation of Gln40 of Nedd8 to glutamate (Q40E) effectively inhibits CRL E3 activities, directly interfering with cellular functions. However, the molecular mechanism by which Nedd8(Q40E) affects CRL function is unclear. Cross‐linking mass spectrometry (XL‐MS) is a powerful strategy that can be utilized to characterize dynamic and heterogeneous protein complexes that are difficult to study using conventional structural tools. Quantitative cross‐linking mass spectrometry (QXL‐MS) not only allows the determination of physical contacts between and within proteins at the amino acid resolution, but also enables quantitative assessment of conformational changes in protein complexes in various states. To elucidate the structural basis underlying the activation/inactivation of CRLs through neddylation, we have employed novel QXL‐MS strategies to compare cross‐link profiles of different types of full‐length human Cul1‐Rbx1 complexes, as well as their interactions with E2‐conjugating enzyme Cdc34. Thus far we have uncovered major Nedd8‐induced structural rearrangements of the CRL1 catalytic core that are not induced by Nedd8(Q40E) conjugation, suggesting that the subtle change of a single Nedd8 amino acid is sufficient to re‐orient the topology of the CRL catalytic core. Our results provide new insights into how neddylation regulates the conformation and activity of CRLs.

Support or Funding Information

This work is supported by NIH RO1GM074830 to L.H, and R01GM106003 to L.H. and S. R..
Decreased Aggregation of Carboxypeptidase O upon Incorporation into Lipid Rafts
Authors: Ezeribe Hazel O , Burke Linnea C , Barfi Gifty B , Lyons Peter J
Published: November 2023

Carboxypeptidase O (CPO) is a member of the M14 family of proteolytic enzymes. Many members of this family are secreted from cells and are involved in degradation of extracellular peptides; other carboxypeptidases remain within the secretory pathway where they are involved in the maturation of bioactive peptides. CPO is a GPI‐anchored carboxypeptidase that is expressed in epithelial cells of the small intestine. To further understand the function of CPO, we investigated its subcellular localization. CPO was found to be present in detergent‐insoluble complexes, suggesting a lipid raft localization and function. Immunofluorescence analysis of CPO in stably‐transfected MDCK cells, however, indicated a punctate distribution of aggregates having little co‐localization with lipid raft proteins. Incubation with cholesterol dramatically decreased this aggregation and resulted in the localization of CPO to puncta of uniform size (< 300nm diameter) and distribution. Similar results were obtained when cells were incubated with oleic acid, which also led to increased GPI anchoring and membrane association. We reasoned that increased membrane cholesterol might mediate conformational changes in CPO, via its GPI anchor, that result in decreased aggregation. We found that loss of the GPI anchor by deletion of the C‐terminal GPI signal peptide, but not the N‐terminal partial prodomain, resulted in greatly reduced expression of CPO in Sf9 cells, suggestive of a conformational and/or folding defect. A decrease in expression of this C‐terminally deleted CPO was also observed in HEK293T cells, although further analysis showed no alteration in protease susceptibility of CPO that might suggest large changes in conformation or folding. Our results suggest that incorporation of CPO into cholesterol‐rich lipid rafts leads to subtle changes in CPO conformation that prevent aggregation.

Support or Funding Information

Supported by Faculty Research Grants to PJL from Andrews University
Exploring the Evolution of a Unique Allosteric Regulatory Mechanism in Caspase‐6
Authors: Grinshpon Robert D. , Clark A. Clay
Published: November 2023

Caspase‐6 selectively degenerates excess axons during brain development, and then activity is diminished in healthy adult brains. However, it is found expressed and active in all stages of Alzheimer's Disease (AD), so it has been hypothesized to drive AD pathology. However, highly specific caspase inhibitors are necessary for meaningful experimentation, but remain elusive. Caspase active site inhibitors exhibit cross‐reactivity due to structural conservation of the caspase family's active site, which has lead to inconclusive experimental results. We are exploring allosteric options by studying helix‐3 of caspase‐6, in which twelve N‐terminal residues undergo a strand‐to‐helix conformational change that renders the enzyme inactive when zinc binds to a unique exosite. We used a helical propensity prediction algorithm (AGADIR) to design two mutants that prevent the transition; (1) locked in the active canonical conformation, and (2) locked in the inhibited extended helix conformation (EHC). For the first mutant, we redesigned helix‐3 of caspase‐6 to mimic the propensity seen in caspase‐3, because other capases cannot undergo the transition. We found that only two out of the eighteen residues in the extended helix region are conserved, and that only two mutations were necessary to restore the helical propensity found caspase‐3. We will also explore the access to the EHC in other caspases in order to glean information about how the new function of helix‐3 evolved. Our lab's goal is to learn about the intricacies of caspase allosteric regulation in order to bypass the active‐site conservation dilemma. We will then exploit these naturally existing regulatory mechanisms for the advancement of human disease research.
Brain Expressed X‐linked 3 (BEX3) is a High‐Order Oligomer with Partially Folded Regions
Authors: Cabral Katia , Raymundo Diana , Silva Viviane , Sampaio Laura , Hill Luis Fernando , Almeida Fabio , Cordeiro Yraima , Almeida Marcius
Published: November 2023

The members of the family of Brain Expressed X‐linked (BEX) are mammal‐specific proteins involved in neurodegeneration, cell cycle and cancer. However, very limited data are available on the conformational state of any member of the BEX family. Recently, BEX3 has been predicted to be intrinsically disordered and also to represent an intracellular hub for cell signaling. In this study, we characterized the BEX3 structure using biophysical experimental data. The association of atomic force microscopy and small angle X‐ray scattering revealed that BEX3 forms a specific higher‐order oligomer that is consistent with a globular molecule. The partial proteinase K digestion has suggested that BEX3 structure contains a core which is proteolysis‐resistant around residues 55–120 with unfolded regions at the N‐ and C‐termini. Circular dichroism spectroscopy and solution nuclear magnetic resonance data revealed that BEX3 exists in solution as an oligomer with partially disordered segments at the N‐ and C‐termini and with a more compact helical structure in a central core. The self‐oligomerization of BEX3 has been previously reported in cell culture and is consistent with our in vitro data. We speculate whether the self‐association of BEX3 into a folded core offers a natural mechanism to protect it from degradation and possibly even to regulate its activity.

Support or Funding Information

FAPERJ, CNPq, INBEB, CENABIO.
Hydrodynamic Radius Prediction of Intrinsically Disordered Proteins
Authors: Ricard Benjamin , Tomasso Maria , Whitten Steven
Published: November 2023

Intrinsically disordered proteins are proteins that do not show a set three‐dimensional tertiary structure and are usually involved in signal‐transduction and transcription regulation. It is estimated that upwards of 33% of all eukaryotic proteins contain intrinsically disordered domains. It is known that hydrodynamic radius (Rh) of IDPs depends largely on their intrinsic propensity to conform into a polyproline‐II helix. A generalized understanding of the molecular organization of IDPs in response to perturbations is presented here. This model uses polyproline‐II propensities and net charge in order to simulate Rh and understand the structural properties of IDPs in terms of discrete changes in a system. Fundamental understanding of protein structures in IDPs may provide an avenue for novel drug design, diagnostics, and treatment.

Support or Funding Information

Research reported in this poster was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award R15GM115603.
Elucidating ER‐Associated Degradation Pathway for GABAA Receptors
Authors: Mu Tingwei
Published: November 2023

Proteostasis maintenance of g‐aminobutyric acid type A (GABAA) receptors dictates their function in controlling neuronal inhibition in mammalian central nervous systems. However, as a multi‐subunit, multi‐span integral membrane protein, even wild type subunits of GABAA receptors fold and assemble inefficiently in the endoplasmic reticulum (ER). Moreover, numerous point mutations in GABAA receptors aggravate such inefficient processes, leading to loss of their function and thus epilepsies. Unassembled and misfolded subunits undergo ER‐associated degradation (ERAD), but this degradation process remains poorly understood for GABAA receptors. Here, using the a1 subunits of GABAA receptors as a model substrate, we demonstrated a Hrd1‐mediated ubiquitination and VCP‐mediated extraction pathway. Repressing the initial ERAD recognition step enhanced the functional surface expression of misfolding‐prone a1(A322D) subunits, which causes autosomal dominant juvenile myoclonic epilepsy. This study clarifies an ERAD pathway for GABAA receptors, providing a novel way to finely tune their function in physiological and pathophysiological conditions.
TRANSLATIONAL REGULATION OF HYPOXIA‐INDUCIBLE FACTOR 2α IN RESPONSE TO IRON AND CELLULAR STRESSORS
Authors: Lin Willey , Deck Kathryn , Eisenstein Richard
Published: November 2023

Hypoxia‐inducible factor 2α (HIF‐2α) is a heterodimeric helix‐loop‐helix transcription factor that plays an integral role in the regulation of erythropoiesis and iron metabolism and is a key player in the oxygen‐sensing and signaling pathways. HIF‐2α also stimulates lipid synthesis and storage in liver suggesting a role in controlling fuel metabolism in hypoxia and iron deficiency. HIF‐2α's regulatory role in iron metabolism makes it a subject of interest in relation to blood disorders, in particular anemia induced by iron deficiency or inflammatory disorders. HIF‐2α expression has been thought to be primarily controlled via oxygen‐ and iron‐dependent degradation by the action of prolyl hydroxylases and the von Hippel Lindau (VHL) E3 ubiquitin ligase. However, our lab and others have shown that dysregulation of HIF‐2α mRNA translation leads to hematological disorders. We are interested in examining the controls of HIF‐2α mRNA translation in response to iron and the presence of different stressors including the inflammation associated with diet‐induced obesity. Our research currently focuses on the 5′ untranslated region (UTR) of the mRNA of HIF‐2α. In the 5′ UTR of HIF‐2α mRNA is an inhibitory regulatory element called the iron responsive element (IRE). Iron regulatory proteins (IRP) bind to the IRE and are important regulators of HIF‐2α mRNA translation. The IRE impedes translation when bound to IRP as during iron deficiency. When iron levels are elevated, the IRP is inactivated and HIF‐2α mRNA is translated.

To study translational regulation of HIF‐2α we created a reporter construct with the HIF‐2α 5′ UTR linked to GFP. Our plasmid, obtained from R. Tsien, also encodes RFP to control for transfection efficiency. Constructs containing the full length and truncated versions (all lacking the IRE) of the HIF‐2α 5′ UTR were used to investigate the role of the IRE and possibly other translational regulatory elements. Plasmids were transfected into HEK 293 cells and subjected to iron in the form of hemin (H), or an iron chelator, desferal (D). The wildtype HIF‐2α 5′UTR‐GFP construct was induced by hemin and repressed by desferal with a H/D ratio of 2.6. The deletion mutant with the shortest 5′ UTR (80% deletion) directed more than a 2‐fold higher level of GFP activity compared the wildtype and other truncation mutants indicating the presence of a negative regulatory element that is 3′ of the IRE. Surprisingly, all deletion mutants exhibited modest (~1.4 fold H/D ratio) response to iron suggesting the presence of additional regulatory elements responsive to iron that are independent of the IRE. Additional studies are underway to examine the translational regulation of HIF‐2α in animals.

Support or Funding Information

Support NIH DK66600
Achieving Tenure as a Clinical Researcher
Authors: Foreman K. Bo
Published: November 2023

On average, a tenure track position at a four‐year college typically consists of a seven‐year probationary period. In a recent survey, approximately 20% of faculty are denied tenure resulting in loss of employment. To achieve tenure, faculty are typically required to meet specific benchmarks in research, teaching, and service. Meeting these benchmarks is often challenging. Therefore, the purpose of this talk is to address some of these challenges, specifically related to benchmarks in research. This talk will be structured around implementing a focused line of research, discussing the importance of infrastructure, and understanding the expectations of the institution.

A seven‐year probation period may sound like a considerable amount of time to implement a focused line of research; however, this time passes quickly. Even with a focused line of research from a postdoctoral fellowship or doctoral research experience, new tenure track faculty members may face the challenge of implementing their research in a new institution and acquiring funding to support their research. To overcome this obstacle, developing strong collaborations with clinical faculty and/or clinical environments who share similar research goals can lead to subject recruitment, referrals, research ideas, and funding.

Appropriate infrastructure is also critical and should incorporate both the physical structure and institutional support necessary to be successful. Depending on the line of research, typically the physical structure includes space, equipment, and associated startup funding. Physical structure is critical to have in place to be able to focus on research and scholarly productivity without having to worry about procuring space and equipment. In addition to the physical structure, institutional support must also be considered which includes items such as structured mentorship, educational opportunities, grant resources, statistical resources, and accounting support to name a few. Both physical structure and institutional support are critical to acquiring funding to keep a research program viable and productive.

Lastly, understanding the expectations of the institution is important and often incorporates multiple levels of evaluation. Evaluation typically is performed at the department, college, and university levels. Because of this, faculty members need to make sure their focus is in line with the mission of their department, college, and university and firmly valued by the administration. This can be accomplished through initial interviews, yearly formal and/or informal reviews, peer evaluations, and frequent discussions with colleagues, mentors, and supervisors.

In summary, this presentation will provide insight into some of the successes and pitfalls faced in achieving tenure as a clinical researcher.
Computational Design of Helical Peptides Targeting Transmembrane Domain of Cellular Receptors
Authors: Hu Hailin , Zhang Shao‐Qing , DeGrado William Frank
Published: November 2023

Computed helical anti‐membrane protein (CHAMP) design has been shown to be a successful strategy to generate specific binding partners of αIIb and αv integrin transmembrane (TM) domains. In this research, we attempt to extend this method to new transmembrane receptor targets. Our first focus is the design of an antiparallel CHAMP for the mouse Erythropoietin Receptor (EpoR) transmembrane domain, which displays a typical sequence pattern of Ala Coil (Small‐X6‐Small motif). Searching this motif in the sequence space in our previously generated transmembrane helix dimer database results in several templates with optimal geometry as a starting point for design. Thiol exchange and Flash‐EDT assays demonstrate that the designed EpoR‐CHAMP interaction has a comparable affinity as in EpoR TM domain homodimerization. Our results demonstrate the strength of CHAMP design strategy and will probably generate a potent EpoR antagonist for biological research. Furthermore, we are also exploring to target other growth factors, like EGFR family receptors and other integrin members using this method.

Support or Funding Information

This work is supported by NIH grant GM54616. Hailin Hu is supported by China Scholarship Council.
Juncture of the cytoskeleton and innate immune signaling pathways: the role of Suppressor of IKK epsilon (SIKE)
Authors: Dohleman Emerald L , Steele Hannah J , Knoblauch Jilan B , Sonnenschein Halie A , Lawrence Kenneth F , Bell Jessica K
Published: November 2023

The innate immune system rapidly responds to challenges by pathogens via activation of an inflammatory response. As a convergence point for multiple inflammatory and anti‐viral signaling pathways, TANK binding kinase 1 (TBK1) serves as a catalytic hub to initiate host defenses. Suppressor of IKK epsilon (SIKE) is a newly identified TBK1 substrate. The goal of this project was to identify the function of SIKE within the host's anti‐viral response. Co‐immunoprecipitation – tandem MS/MS analyses of the SIKE interaction network identified several interactions with cytoskeletal components. Immunofluorescence assays of endogenous SIKE with cytoskeletal markers and colocalization analyses indicated that SIKE colocalized with tubulin, actin, and a‐actinin. Reciprocal immunoprecipitation (RcIPs) studies confirmed the SIKE:tubulin and SIKE:a‐actinin interaction that appeared enhanced following dsRNA stimulation, a mimic of viral infection. ELISA assays were used to characterize the binding affinity between SIKE and the cytoskeletal proteins under unstimulated and simulated viral‐stimulated conditions. The SIKE:actin interaction was not detected by RcIP. In vitro immunoprecipitation assays mapped direct interactions between SIKE:tubulin and SIKE:a‐actinin and were used to further probe for a weak SIKE:actin interaction. Assays employed purified cytoskeletal proteins and full‐length (residues 1–207), N‐terminal (residues 1–112), C – terminal (residues 113–207) or a phosphomimetic (S6E) of a 6x‐His‐tagged SIKE construct. Together these studies establish an interaction between SIKE and cytoskeletal proteins that may provide a direct link between innate immune signaling and cytoskeletal components.

Support or Funding Information

This work was supported by NIH grant R21AI107447 and the University of San Diego SURE program.
Probing the Subunit Interface of Malate Dehydrogenase: Effects of S266A and L269A Mutations
Authors: James Nikia , Verdi Jayme , Bell Ellis , Bell Jessica
Published: November 2023

Malate Dehydrogenase is thought to involve a reciprocating subunit mechanism involving ligand induced interactions across the subunit interface in the dimer and such a mechanism is thought to be involved in both catalysis and citrate regulation of this important enzyme. Clustal analysis of organelle forms of the enzyme and HINT analysis of interactions across the interface have identified two residues, S266 and L269 that could be involved in communication between the active site and the subunit interface. Two mutants, S266A and L269A have been constructed, expressed and purified by Nickel‐NTA affinity chromatography. Initial rate kinetic studies of each mutant reveal unique non‐ Michaelis‐Menten behavior. S266A shows linear Michaelis menten plots with Oxaloacetate as varied substrate but normal saturation with NADH while L269A shows non‐Michaelis Menten behavior with NADH as varied substrate but normal saturation with Oxaloacetate. To construct a heterodimer with one subunit containing the mutant while the other subunit is normal at the interface but unable to effectively bind Oxaloacetate as a result of mutation of upto three of the active site arginines, we have explored reversible dissociation of each relevant homo dimer to create heterodimers. Preliminary results suggest that while heterodimers can be separated by ion exchange chromatography the dynamic nature of the dimer formation creates problems. To overcome this complication we are exploring chemical cross‐linking of the dimer form of the enzyme with bifunctional imidates.
Noncanonical PDZ Interaction between PKA RIα R368X Acrodysostosis Mutant and P‐Rex1 PDZ Domain
Authors: Del Rio Jason C , Bruystens Jessica G.H. , Chávez‐Vargas Lydia , Ma Yuliang , Kornev Alexandr P. , Vázquez‐Prado José , Taylor Susan S.
Published: November 2023

Protein Kinase A (PKA), a tetrameric enzyme comprised of two catalytic (C) and two regulatory (R) subunits, is the prototypical kinase that responds to intracellular cAMP. Hindering the capacity of the RIα subunit isoform to bind cAMP, results in overregulation of the C subunit, a rare condition called Acrodysostosis (ACRDYS). Patients with ACRDYS typically have mutations in either RIα or PDE4D genes, and display stunted growth, mental retardation, and in some cases, hormonal resistance. Recently, a new interaction between RIα and the tandem PDZ domains of Phosphatidylinositol 3,4,5‐Triphosphate‐Dependent Rac Exchanger 1 (P‐Rex1) was discovered. P‐Rex1, a multi‐domain scaffolding protein with Rac specific guanine nucleotide exchange factor (GEF) activity, is highly expressed in certain leukocyte populations, and is intimately involved with regulation of cell migration. Using peptide blots, we scanned the Tandem PDZ domains of P‐Rex1 (residues 607–783) for interactions with RIα. We found that RIα bound to the α2 helix and the linker region between the tandem PDZ domains. Pulldown assays from HEK293 cells further confiirmed the interaction of RIα and P‐Rex1 PDZ domains. Interestingly, an ACRDYS mutation of RIα (R368X), deleting the last 14 amino acids from the C‐terminus of RIα, bound stronger to the PDZ domains of P‐Rex1 in contrast to the canonically defined mechanism of PDZ domain interactions, which bind to and recognize the C‐terminus via a β‐strand addition mechanism. Having solved the structure of this R368X ACRDYS mutant allowed us to model this noncanonical complex in silico using ZDock. We found that the PDZ domain may bind in the pocket of the second cyclic nucleotide binding domain (CNB‐B) of RIα, in agreement with our peptide blot data. This suggest that Acrodysostosis may not only alter regulation of the C subunit, but also change the PKA signalosome. We hope to validate this interaction by X‐ray crystallography.

Support or Funding Information

This Work was supported by NIH Grant GM34921 to Susan S. Taylor.

Jason C. Del Rio was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences, T32 GM007752.