bluuebunny/crossref_metadata_2025 huggingface dataset:
Conformational dynamics of SNARE recycling mediated by NSF
Authors: Choi Ucheor B. , Dunleavy Katie , Matlock Holland , Gething Claire , Howells Giovanni , Misra Bishal , White K I. , Brunger Axel
Published: May 2022

Neurotransmitter release via synaptic vesicle exocytosis is mediated by the dynamic assembly and disassembly of the neuronal SNARE (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor) complex, which consists of syntaxin‐1, SNAP‐25, and synaptobrevin‐2. Despite their importance, the molecular mechanism of SNARE complex recycling remains unclear. Individual SNARE proteins are intrinsically disordered and undergo a disorder‐to‐order transition, which assembles into a highly stable four‐helix bundle, providing the energy required to drive membrane fusion between the synaptic vesicle and the plasma membrane. The AAA+ protein NSF later disassembles the SNARE complex to maintain a pool of the individually functional SNARE proteins to be utilized for recurring rounds of synaptic vesicle fusion. Using single‐molecule fluorescence resonance energy transfer (smFRET), we examined the stepwise conformational dynamics of individual SNARE proteins during NSF‐mediated disassembly and reassembly of the SNARE complex. Interestingly, the disorder‐to‐order transition of the SNARE proteins during SNARE complex assembly was reversible by NSF‐mediated disassembly, where the SNARE chaperone Munc18 preserved the intrinsically disordered state of SNAP‐25 and synaptobrevin‐2 by locking syntaxin‐1 in an inhibiting closed conformation. Moreover, we observed a transient “entangled” conformation of SNAP‐25 during the reassembly process of the SNARE complex. Together, NSF acts as a protein quality control mechanism for efficient membrane fusion via proper assembly of the SNARE complex.
Detection of Multi‐Protein Complexes Containing PCNA Using Fluorescence Anisotropy and Hormetic Modeling
Authors: Greenwood Sharon N. , Belz Regina G. , Weiser Brian P.
Published: May 2022

Proliferating Cell Nuclear Antigen (PCNA) is a homotrimeric protein involved in DNA replication and repair. It contains three equivalent binding sites that are known to interact with dozens of replication and repair proteins. In theory, PCNA could bind multiple proteins at the same time, but steric hindrance prevents this for some protein combinations. Here, we developed an approach to detect the formation of multi‐protein complexes containing PCNA and base excision repair proteins using fluorescence anisotropy and hormetic modeling. Initially, we measured binding of fluorescent‐labeled pogo‐ligase peptide (PL) to PCNA and determined a Kdof 118 nM. This assay measured an increase in the fluorescence anisotropy of 50 nM PL upon PCNA binding because the fluorescent peptide becomes part of a larger macromolecular complex that slows its movement in solution (anisotropy min/max: 0.043/0.121). Next, we competed 50 nM PL from 0.25 μM PCNA using uracil DNA glycosylase (UNG2), which reduced the anisotropy from 0.086 to 0.057, and we determined an IC50 of 4 μM with a conventional sigmoidal curve. In contrast, displacement of PL from 3 μM PCNA using UNG2 resulted in a hormetic dose response that was fit using the Brain‐Cousens equation for hormesis. In these experiments, the anisotropy of 50 nM PL increased from 0.095 in the presence of 3 μM PCNA to 0.127 with the addition of 5 μM UNG2, and the anisotropy then reduced to 0.086 with 50 μM UNG2. The hormetic response occurred because a ternary PCNA‐PL‐UNG2 complex formed at UNG2 concentrations that were sufficient to bind open PCNA sites, but were insufficient to displace PL from PCNA, and the anisotropy of PL bound to the ternary complex was higher than its anisotropy bound to PCNA alone. Additional values derived from the Brain‐Cousens curve included a significant hormesis parameter f (0.024), a maximum stimulatory response of 134% of control at a dose of 4 μM UNG2, and a limited dose for stimulation at 31 μM UNG2. We also report additional datasets where UNG2 displaced 50 nM PL from other PCNA concentrations to train our hormetic modeling and optimize assay signal/noise. Continuing experiments using fluorescent‐labeled UNG2 and fluorescent‐labeled DNA polymerase β (POLB), in addition to DNA Ligase 1 (LIG1), will explore the simultaneous and/or sequential interactions of base excision repair proteins with PCNA.
Genomic Study of Histamine Receptor Ligand Binding Sites of the Bivalve Mollusc Crassostrea virginica
Authors: Mansfield Kera J. , Wallach Rosanne , Catapane Edward J. , Hinkley Craig , Carroll Margaret A.
Published: May 2022

Histamine is a biogenic amine found in a wide variety of invertebrates. Histamine is particularly well studied in arthropods and gastropods where it is involved in local immune responses as well as regulating physiological functions in the gut. Histamine also functions as a neurotransmitter, especially for sensory systems. Previous physiology work of our lab found that histamine activates the sensory system of Crassostraea virginica, eliciting a motor response in the gill. Our earlier cell biology and immunofluorescence work also showed the presence of histamine receptors in ganglia and mantle of C. virginica. Recently the genome of C. virginicaand other bivalves have begun to be mapped. By conducting BLAST searches of the NCBI (National Center for Biotechnology Information) database using DNA and protein sequences of C. virginica and other invertebrate and mammalian species we found matches for histamine receptor H1R genes on chromosome 8; H2R on chromosomes 1, 2, 5 and 10; and H3R on chromosome 3. BLASTS of other invertebrates and mammals found matches with very low Expect Values (E Values) and moderately high Percent Identity, signifying similarities of H1R, H2R and H3R of C. virginica to those of other bivalves, gastropods, insects, mice, rats and humans. We hypothesize that the ligand binding sites (LBS) for H1R, H2R and H3R receptors in C. virginica are evolutionarily conserved and will closely match those of other animals. To study this, we conducted searches of the NCBI database for H1R, H2R and H3R receptors LBS of C. virginica and compared them to other animals. We found the LBS for H2R in C. virginica was identified and match some other invertebrates well, but did not match humans of other mammals very well. The LBS for H3R matched some other bivalves, invertebrates as well as humans and other mammals well. The LBS for H1R in C. virginica and other invertebrates we looked at has not yet been identified. The LBS for H1R in humans and other mammals is very highly conserved. This study complements our earlier physiology and cell biology studies demonstrating the presence and function for histamine in C. virginica, and shows that the genome of C. virginica contains genes to produce histamine receptor LBS that are similar to those of other animals where it has been identified. This new information is valuable as it shows that the simple nervous system of histamine can be used to expand studies on histamine neurotransmission.
Effects of RTI‐567 on the Neuropeptide S System
Authors: Jewula Gabriel J. , Runyon Scott , Clark Stewart
Published: May 2022

Anxiety and post‐traumatic stress have increasingly become more prominent disorders and options for treatment remain limited. One of the most prescribed therapeutics are benzodiazepines. Unfortunately, these anxiolytics have negative side effects and are not as effective in the long term. The recently discovered Neuropeptide S (NPS) system may lead to an alternative, potentially improved, line of therapeutics. This system consists of a known endogenous neuropeptide, Neuropeptide S and a G‐protein‐coupled receptor termed the Neuropeptide S receptor (NPSR). Activation of this receptor leads to an increase in intracellular calcium and a mobilization of cAMP. This is associated with behavioral phenotypes such as anxiolysis, enhanced memory consolidation, and hyperlocomotion. The NPSR is highly expressed in the amygdala linking the NPS system with fear and anxiety. Previously, as part of our ongoing drug discovery program, a biased agonist (RTI‐263) was identified that preferentially enhances the calcium signaling pathway over the cAMP pathway following NPSR binding. This biased agonist also reduced hyperlocomotion or arousal observed with NPS, while maintaining the anxiolytic component. In the present study we test another compound (RTI‐567) using classic anxiolytic, locomotor, and memory testing paradigms such as the light dark box, open field, and inhibitory avoidance. Our overall goal is to identify novel synthetic agonists with reduced side effects and improved long‐term efficacy.
Using Controlled Environment Agriculture as a Platform for STEM‐Training
Authors: Johnson Tabetha , Payne Marvin A.
Published: May 2022

The purpose of this summer STEM Bridge initiative was to provide incoming first‐year university students with interdisciplinary STEM training using a Controlled Environment Agriculture hydroponic container farming tech platform coupled with soft skills and increased early access to the campus facilities. A pilot was conducted during the summer of 2021 on the La Sierra University campus. The two‐week collaborative experience included faculty from eight academic units including from Science, Humanities, and Business. We offered students experiments and projects from hydroponic basics, use of sensors and computer control systems, to developing sales presentations and business models. This approach introduced students to career pathways– many within STEM, centered around a tech platform capable of contributing to real‐world solutions to long‐standing community problems. We also emphasized the importance of inclusive representation in STEM, the importance of access, and of including all voices. Students developed a meaningful peer and faculty support system and as an additional benefit became familiar with how to navigate the campus culture. Preliminary results indicate the students felt their foundational understanding of the sciences improved after attending STEM Bridge. A portion of the students indicated a newly acquired desire to obtain a terminal STEM degree after attending STEM Bridge. Most of the students felt an increase in awareness of resources on campus to support them academically and knowledge of their way around campus. All students felt an increase in preparedness for the academic rigors of college life while most students reported feeling an increase in their ability to manage their time well after the program.
SARM1 NAD Hydrolase Deficiency Normalizes Fibrosis and Ameliorates Cardiac Dysfunction in Diabetic Hearts
Authors: Lee Chi Fung , Nizami Hina , Gu Haiwei , Light Christine
Published: May 2022

Decline in cellular NAD levels emerges as a hallmark of diseases such as diabetes and heart failure. Strategies to activate NAD synthesis pathways and replenish NAD levels have shown promises to treat heart disease. However, how NAD consumption mechanisms lead to NAD depletion and disease pathogeneses are less understood. SARM1 is an intracellular NAD hydrolase, and its role in heart disease has never been investigated. We therefore hypothesized that SARM1 deficiency may protect hearts against diabetic cardiomyopathy. Male C57BL/6 wild‐type (WT) and SARM1‐KO mice were challenged with 16‐week diabetic stress initiated by streptozotocin injections (STZ). Longitudinal cardiac function was measured by echocardiography after 8 or 16 weeks of diabetes, and tissue and plasma samples were harvested for biochemical and molecular analyses. Chronic diabetic stress lowered cardiac NAD levels and led to progressive decline in systolic function of WT mice. Diabetic SARM1‐KO mice showed improved systolic function and increased cardiac NAD levels, compared to diabetic WT mice. Progressive decline in diastolic function induced by chronic diabetes was also improved by SARM1 deficiency. Heart weights and hyperglycemia were similar in diabetic WT or diabetic SARM1‐KO mice. Metabolomic analyses of plasma showed significant changes in 58 out of ~300 aqueous metabolites assessed when comparing non‐diabetic WT to diabetic WT plasma, including increased levels of glucose and branched‐chain amino acids. However, the 58 metabolites showed no changes in levels when comparing diabetic WT to diabetic SARM1‐KO plasma. Similarly, we found that 26 out of 85 lipid species showed significant changes when comparing non‐diabetic WT to diabetic WT plasma, and only one of these 26 lipid metabolites showed a significant change between diabetic WT and diabetic SARM1‐KO plasma. The data suggest that cardioprotection in SARM1‐KO mice was likely due to the reversal of cardiac pathogenic mechanisms but not improved systemic metabolic state.

To determine how SARM1 deficiency affects cardiac NAD metabolism, expression of genes in NAD metabolic pathways were assessed. Levels of Bst1 and Cd38 did not change in SARM1‐KO hearts, suggesting that SARM1 deficiency did not induce compensatory changes in expression of the other NAD hydrolases. SARM1‐KO hearts showed lower expressions in some of the NAD consuming genes (e.g. Sirtuins) and NAD synthesis genes (e.g. Nmnats). Cardiac dysfunction marker, Nppb expression, was induced in diabetic WT hearts, and was reversed by SARM1 deficiency. Cardiac fibrosis and pro‐fibrotic genes (e.g. ctgf) were induced in diabetic WT hearts and were suppressed by SARM1 deficiency. Using the total SARM1 KO mice, our data support that SARM1 deficiency normalizes decline in NAD levels and pro‐fibrotic pathways to ameliorate diabetic cardiomyopathy. Further experiments are warranted to dissect the cell‐type specific roles of SARM1 in diabetic hearts.
Transcriptional regulation via strand displacement DNA repair in G‐quadruplexes
Authors: Whitaker Amy , Freudenthal Bret
Published: May 2022

Environmental exposures and lifestyle choices can result in cellular oxidative stress, characterized by the generation of an abundance of reactive oxygen species (ROS). ROS wreak havoc on the structure of DNA bases, with guanine modification yielding the lesion 8‐oxo‐7,8‐ dihydroguanine (8oxoG) being particularly prevalent. If not repaired, 8oxoG is mutagenic, causing G to T transversion mutations that can initiate and promote human disease. Guanine‐rich G‐ quadruplex (G4) forming sequences are enriched at promoter proximal regions of the genome, making these regions hot spots for 8oxoG lesions. The cells primary defense against 8oxoG is base excision repair (BER). Recently, it was shown that the repair of 8oxoG by the BER enzymes OGG1 and APE1 perturbs the structural equilibrium of the VEGFpromoter DNA sequence between duplex and G4 conformations, resulting in epigenetic‐like modifications of gene expression. However, the mechanistic details regulating this equilibrium remain somewhat enigmatic, including the activity and coordination of BER enzymes on an 8oxoG containing G4 promoter. To address this, we determined the requirement and efficiency of each BER factor on G4 substates (including OGG1, APE1, Polβ, and DNA ligase 1) by employing a combination of pre‐steady state kinetics assays and in vitroBER reconstitution assays with G4 DNA substrates. Surprisingly, we observe OGG1 is capable of initiating BER on oxidized G4 VEGFDNA substrates in which the G4 region is flanked by non‐G4, duplex regions of DNA. Pre‐steady state kinetics revealed that compared to abasic duplex DNA, APE1‐mediated strand cleavage is only slightly decreased and that a majority of the reduction in its activity on G4 DNA results from slow product release. Thus, APE1 may have an opportunity to recruit transcription factors to the VEGF promoter during this slow product release step of its reaction. Interestingly, Polβ performs multiple insertions on G4 substates via strand displacement DNA synthesis, with 3 ‐ 5 insertions depending on the position of damage. In contrast, there is only a single insertion by Polβ with corresponding duplex DNA substates and no insertion when the G4 structure lacks an opposing strand with flanking duplex regions. Ligase I does not efficiently ligate G4 substrates, and hence cannot complete the BER repair cycle. Instead, proper repair requires the long‐patch BER enzyme flap‐endonuclease activity of FEN1 in response to the multiple insertions by Polβ prior to ligation.
Rho5 regulates lysophosphatidic acid induced macropinocytosis in Entamoeba histolytica
Authors: Datta Sunando , Apte Achala , Manich Maria , Labruyère Elisabeth
Published: May 2022

Entamoeba histolytica is a unicellular enteric protozoan responsible for variety of gastrointestinal pathologies. The pathogenicity of the parasite is attributed to its dynamic cytoskeleton. Rho family GTPases, a major class of molecular switches, play an indispensable role in controlling cytoskeleton remodelling. Rho GTPases are known to be activated by specific Guanine nucleotide Exchange Factors (GEF) upon stimulation by various extracellular stimuli to regulate signalling.

We have established that EhRho5 is important for LPA induced micropinocytosis in the amoebic trophozoites. EhRho5 translocated from cytosol to plasma membrane and endomembranous compartments when stimulated with LPA, a bioactive phospholipid known to activate Rho subfamily members (1,2). Our findings were strengthened when FRAP (Fluorescence recovery after photobleaching) study showed that LPA treated cells show 30% faster recovery of Rho5 when compared to serum starved cells. The translocation is accompanied by activation of the GTPase as revealed by effector pull down assay(2). LPA stimulation enhanced the macropinocytic efficiency of trophozoites in a Rho5 dependent manner. We then identified the GEF for Rho5 which too, shows translocation upon stimulation with LPA. Also, depletion of the GEF leads to decreased pinocytic efficiency of the trophozoites. Using inhibitor based studies, we further demonstrated that PI3K acts as an intermediate cue for EhRho5 activation during LPA induced macropinocytosis.

Moreoever, Rho5 expression showed contrary relationship with random migration of the trophozoites suggesting a pivotal role of the GTPase in mutally antagonistic cellular processes(3) in the amoeba.

Reference:

Fleming IN, Elliott CM, Exton JH. Differential Translocation of Rho Family GTPases by Lysophosphatidic Acid, Endothelin‐1, and Platelet‐derived Growth Factor, J. Biol. Chem. 1996, 271: 33067–33073

Van Leeuwen FN, Olivo C, Grivell S, Giepmans BNG, Collard JG, Moolenaar WH. Rac Activation by Lysophosphatidic Acid LPA1 Receptors through the Guanine Nucleotide Exchange Factor Tiam1, J. Biol. Chem. 2003, 278:400–406

Veltman DM, Lemieux MG, Knecht DA, Insall RH. PIP3‐dependent macropinocytosis is incompatible with chemotaxis. Journal of Cell Biology. 2014 Feb 17;204(4):497‐505.
Ligand‐specific mechanisms of allosteric regulation in FXR
Authors: Yu Tracy
Published: May 2022

Farnesoid X receptor (FXR) is a member of the nuclear receptor family of ligand‐regulated transcription factors that regulates bile acid, lipid and glucose metabolism. Bile acids are the endogenous FXR ligands. Bile acid binding to FXR induces association with FXR response element sequences (FXREs), and promotes selective recruitment of coregulator proteins via induced structural changes in the activation function AF‐2 surface, i.e. the site of coregulator binding. However, there is a poor understanding of how FXR is allosterically regulated by bile acid, which acts as a molecular switch to initiate critical long‐range of transcriptional events. The goal of this work is to understand how minor structural modifications in bile acids give rise to differential FXR transcriptional activity. We used dual luciferase assays to determine how bile acids modulate FXR DNA binding preferences for a a wide range of FXREs. We also used molecular dynamics (MD) simulations to study the effect of the bile acids on AF‐2 signaling. We have identified key residues that mediate specific allosteric communication pathways in FXR, and the mutations of the key residues in FXR‐LBD affects bile acid‐specific FXR transcriptional activities, confirming their roles in ligand‐AF‐2 allostery. These findings can further our understanding of how bile acids impact FXR promoter selectivity and coregulator recruitment.
Zinc‐Chelating BET Bromodomain Inhibitors Selectively Accumulate and Affect Gene Expression in Pancreatic β‐Cells
Authors: Jones Lipinski Rachel A. , Stancill Jennifer S. , Wynia‐Smith Sarah L. , Sprague Daniel J. , Nunez Raymundo , Bird Amir , Corbett John A. , Smith Brian C.
Published: May 2022

The islets of Langerhans are a heterogeneous mixture of endocrine (hormone‐secreting) cells in the mammalian pancreas. Of particular interest are the β‐cells responsible for maintaining glucose homeostasis by secreting insulin in response to increased blood glucose. Type 1 diabetes (T1D) is characterized by autoimmune‐mediated destruction of β‐cells, resulting in an inability to produce and release insulin in response to changing glucose levels and subsequent reliance on daily insulin injections. There is currently no cure for T1D. The bromodomain and extra‐terminal domain (BET) family of proteins, comprised of BRD2, BRD3, BRD4 and BRDT, are a druggable family of proteins that can be targeted by pan‐BET bromodomain inhibitors (BETi), including the small molecule (+)‐JQ1. Recent studies have shown that targeting the BET family of proteins with BETi may prevent and treat T1D. However, the systemic use of current generation BETi is associated with off‐target, adverse effects including fatigue, gastrointestinal toxicity and memory loss. Since any treatment for T1D would be widely used in a pediatric patient population, avoiding off‐target effects is imperative. Here, we propose to ameliorate the off‐target effects of BET bromodomain inhibition by targeting BETi directly to pancreatic β‐cells. To achieve this, we exploited the high concentration of Zn2+ ions (~20 mM) present in β‐cells relative to other cells in the body. We report the synthesis of a novel, zinc‐chelating analogue of (+)‐JQ1, [(+)‐JQ1‐DPA], in which the BETi (+)‐JQ1 was conjugated, via an ethylenediamine linker, to dipicolyl amine (DPA). DPA chelates Zn2+ and thus enables (+)‐JQ1 to accumulate in β‐cells. As control compounds we also synthesized (+)‐JQ1‐DBA, a non‐zinc‐chelating analogue of (+)‐JQ1‐DPA, in which (+)‐JQ1 was conjugated to dibenzyl amine (DBA) and (‐)‐JQ1‐DPA, a zinc‐chelating compound that is inactive as a BETi. Molecular modeling and biophysical assays showed that (+)‐JQ1‐DPA and (+)‐JQ1‐DBA, but not (‐)‐JQ1‐DPA, retain binding to both bromodomains of BRD4 in vitro. Cellular assays demonstrated that (+)‐JQ1‐DPA retains efficacy; in particular, (+)‐JQ1‐DPA, attenuated the expression of NF‐ĸB target genes in β‐cells stimulated with the pro‐inflammatory cytokine, IL‐1β, a model for β‐cell destruction. Finally, using islets isolated from the INS1Cre; ROSA26mTmG mouse, an established mouse model that expresses enhanced green fluorescent protein in insulin‐positive cells and mTomato in insulin‐negative cells, we demonstrated that (+)‐JQ1‐DPA affects gene transcription in pancreatic β‐cells but not in other pancreatic cell types. In conclusion, we have synthesized a zinc‐chelating analogue of (+)‐JQ1 that is selectively active in pancreatic β‐cells over other islet cell types, including macrophages and α‐cells.
Developing a COVID‐19 Yeast Oral Vaccine for Low Income Countries
Authors: Brysgel Taylor , Kozub Noah , Bresnahan Christopher , Austriaco Nicanor
Published: May 2022

Despite the scientific ingenuity of their design, traditional SARS‐CoV‐2 mRNA vaccines are expensive and require ultracold freezers for storage that are not available in many parts of the world. This makes them inaccessible to third world countries, and no shelf stable, low‐cost alternative currently exists. In response, we have constructed a novel COVID‐19 vaccine delivery system using the probiotic yeast Saccharomyces boulardii. We engineered a plasmid to express the receptor binding domain of the SARS CoV‐2 spike protein. The yeast mating type pheromone secretion signal was fused to the N‐terminus of the immunogen and the Claudin‐4 targeting sequence of the Clostridium perfringens enterotoxin was fused to the C‐terminus. Yeast cells secreting this construct are currently being tested in an animal model to determine if they can elicit an immune response.
Effects of Cannabidiol in a Caenorhabditis Elegans Amyotrophic Lateral Sclerosis Model
Authors: Cavet Romola , Sostena Juliet , Huang Albert , Hauser Paul , De Luke
Published: May 2022

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by the progressive death of motor neurons. Cannabidiol, the second most prevalent cannabinoid in the Cannabis sativa plant, is a potential therapeutic tool for ALS due to its antioxidant, anti‐inflammatory, and anti‐spasticity effects, as well as its complementary role in treating other neurodegenerative diseases. In SOD1‐G93A murine ALS models, cannabinoids have been shown to slow disease progression, extending lifespan and increasing motor function. However, the effects of specific cannabinoids—including cannabidiol—are yet undefined and their functions slowing disease progression are unknown. To advance this understanding we aim to study the effects of cannabidiol treatment in a Caenorhabditis elegans ALS model: a SOD‐1 mutant transgenic strain with SOD‐1 aggregation in muscular cells. We will use a death assay to measure the lifespan of SOD‐1 mutant C. elegans and cannabidiol‐treated SOD‐1 mutant C. elegans to investigate whether treatment with cannabidiol impacts the lifespan of SOD‐1 mutants. To assess mechanosensation, we will touch C. elegans with sutures of various sizes, based on the Von Frey filaments touch response assay in humans. We will develop a novel computational analysis system to measure C. elegans movement in response to touch. We will then compare the motor response of the SOD‐1 mutant transgenic strain to wild type and study if cannabidiol modulates a possible change in motor response. This study will evaluate the functions of cannabidiol as a potential therapeutic tool in ALS using a SOD‐1 mutant C. elegans model.
Minimal mechanistic component of proteasome activation and prevention of impairment by pathological oligomers
Authors: Chuah Janelle J. , Thibaudeau Tiffany A. , Smith David M.
Published: May 2022

Impairment of proteasomal function has been implicated in neurodegenerative diseases, justifying the need to understand how the proteasome is activated for protein degradation. Here, using biochemical and structural (cryo‐EM) strategies in both archaeal and mammalian proteasomes, we further determine the HbYX(‐motif)‐dependent mechanism of proteasomal activation used by multiple proteasome‐activating complexes including the 19S Particle. We identify multiple proteasome α subunit residues involved in HbYX‐dependent activation, a point mutation that activates the proteasome by partially mimicking a HbYX‐bound state, and conformational changes involved in gate‐opening with a 2.1A structure. Through an iterative process of peptide synthesis, we successfully design a HbYX‐like dipeptide mimetic as a robust tool to elucidate how the motif autonomously activates the proteasome. The mimetic induces near complete gate‐opening at saturating concentration, activating mammalian proteasomal degradation of peptides and proteins. Findings using our peptide mimetic suggest the HbYX‐dependent mechanism requires cooperative binding in at least two intersubunit pockets of the α ring. Collectively, the results presented here unambiguously demonstrate the lone role of the HbYX tyrosine in the allosteric mechanism of proteasome activation and offer proof of concept for the robust potential of HbYX‐like small molecules to activate the proteasome.
Neurological restorative effects of the flavanol (‐)‐epicatechin in a rodent model of Gulf War Illness
Authors: Navarrete‐Yanez Viridiana , Rodriguez‐Castaneda Antonio , Ayala Marcos , Ceballos Guillermo , Ramirez‐Sanchez Israel , Villarreal Francisco
Published: May 2022

Gulf War Illness (GWI) afflicts ~30% of the US military personnel who served in the 1990‐91 Persian Gulf War. Symptoms include cognitive deficits, muscle pain, weakness, exercise intolerance and fatigue which are still present after 30 years in war Veterans. Suspect causal agents include exposure to the compounds pyridostigmine (PB), permetrim (PM) and N,N‐diethyl‐m‐toluamide (DEET) which were used as protectants against nerve gases (PB) and insects. We previously reported a study to model GWI in which young male rats were provided PB, PM and DEET at equivalent human doses and physical restraint (to induce stress) for 3 weeks. In GWI animals, skeletal muscle weight was 35% lower vs. controls, which correlated with decreases in myofiber area, limb strength and treadmill time/distance. In a follow up study, we reported on the beneficial effects of the cacao flavanol (‐)‐epicatechin (epi) provided for 2 weeks by oral gavage on skeletal muscle atrophy and loss of function. In the current study, we wished to explore the potential of Epi to beneficially impact neurological measures of memory including multiple markers of hippocampus structure and function. Male Wistar rats underwent 3 weeks of exposure to either vehicles or DEET, PM, PB and stress. Subgroups of GWI rats (n=7/subgroup) received 15 days of either water (vehicle) or 1 mg/kg/day of Epi treatment by oral gavage and were compared to normal control (n=7). Object recognition tasks were performed to assess effects on memory and hippocampus samples were collected for histological and biochemical analysis. Results demonstrate that Epi treatment yields significant improvements in short and long‐term memory vs. GWI rats yielding values similar to those of controls. The assessment of hippocampus oxidative stress (protein carbonylation) and pro‐inflammatory cytokines levels (ELISA) of IL‐1β, TNF‐α and IFN‐γ (figure) yielded significant increases with GWI that were fully normalized with Epi to become comparable to controls. Significant increases in markers of hippocampus neuroinflammation (Iba1 and GFAP) and cell death (caspase activity) were noted with GWI and were significantly reduced with Epi. The neuronal survival signaling pathways (TREM2, PI3K and AKT) were adversely impacted by GWI and were partially or fully restored by Epi. Multiple markers of mitochondrial function (citrate synthase activity and ATP levels) adversely impacted by GWI and were fully restored by Epi. Thus, in an animal model of GWI, Epi beneficially impacted markers of hippocampus neuroinflammation, oxidative stress, cell survival, neurotoxicity and mitochondrial function. Such effects translated into improvements in functional endpoints associated with memory centers. As per the recognized safety profile of Epi clinical studies are warranted to explore the effects of treatment in Gulf War Veterans.
Activity‐Based Profiling of RNA Modifying Enzymes
Authors: Kleiner Ralph
Published: May 2022

Epitranscriptomic RNA modifications can regulate RNA activity, however there remains a major gap in our understanding of the scope of RNA chemistry present in biological systems, the enzymes responsible for regulating modification levels, and the ultimate function of these modifications in biological processes. To address this gap, we have developed RNA‐mediated activity‐based protein profiling (RNABPP), a chemoproteomic strategy relying upon metabolic RNA labeling with modified nucleoside probes, RNA‐protein enrichment, and quantitative proteomics. We apply RNABPP with fluoropyrimidine nucleosides in human cells in order to investigate RNA pyrimidine‐modifying enzymes acting upon mRNA. In addition to profiling 5‐methylcytidine (m5C) and 5‐methyluridine (m5U) methyltransferase activity on mRNA, we identify a novel mechanism‐based RNA‐protein crosslink between 5‐fluorouridine‐modified RNA and the uncharacterized human dihydrouridine synthase DUS3L. Further, we study the cellular substrates of DUS3L using quantitative nucleoside LC‐MS/MS and catalytic cross‐linking and immunoprecipitation‐based sequencing. Taken together, our work provides a general approach for profiling RNA modifying enzyme activity in vivo, and reveals the existence of new pathways for the epitranscriptomic regulation of RNA behavior in human cells.
Role of Mitochondrial TNAP in Thermogenesis and Obesity
Authors: Sun Yizhi , Spiegelman Bruce M.
Published: May 2022

Adaptive thermogenesis has attracted much attention because of its ability to increase systemic energy expenditure and to counter obesity and diabetes. Thermogenic fat cells use creatine to stimulate futile substrate cycling, dissipating chemical energy as heat; but the molecular basis is unclear. Latest data show that thermogenic fat cells localize tissue‐nonspecific alkaline phosphatase (TNAP) to mitochondria to hydrolyze phosphocreatine, which initiates a futile cycle of creatine dephosphorylation and phosphorylation. TNAP expression is powerfully induced when mice are exposed to cold conditions, and its inhibition in isolated mitochondria leads to a loss of futile creatine cycling. In addition, genetic ablation of TNAP in adipocytes reduces whole‐body energy expenditure and leads to rapid‐onset obesity in mice. These data illustrate the critical role of TNAP as a phosphocreatine phosphatase in the futile creatine cycle. Lastly, TNAP is imported into mitochondria through an untypical pathway and finding the regulators of this process may provide important implications of the controlling of the futile creatine cycling in thermic events.
Increased Interest in Anatomical Donation As A Result of Positive Media Coverage
Authors: Walker Cam M. , Ostervoss Tamara E.
Published: May 2022

A 99‐year old female anatomical donor at Oregon Health & Science University (OHSU) in 2018 had an unusual anomaly: Situs inversus (SI) with levocardia [left‐facing heart]. Her SI and levocardia went mostly unnoted throughout her long life, despite several surgical interventions and five successful pregnancies. After consultation with Ms. Ostervoss (director of OHSU’s body donation program), and the donor family, a poster presentation highlighting this donor was prepared and shown at last year’s American Association of Anatomy meeting at Experimental Biology in Orlando, FL, in April 2019 (Walker and Hankin 2019). Additionally, a press release to coincide with the poster presentation was prepared at OHSU for local media consumption. The donor’s story was distributed by print, radio, and television, worldwide.

Afterward, the Body Donation Program (BDP) at OHSU then experienced a surge in applications for anatomical donation. The number far exceeded equivalent applications to be an anatomical donor in past years. This has had beneficial effects for the BDP, including the ability to more fully support the needs of anatomical education, accommodate requests for tissue used in surgical simulation, residency, and specialized workshop training, and to assist other regional institutions with their need for anatomical donors. In addition, the raised awareness of a rare condition has led to a series of research opportunities that may have implications for those who have SI.

The benefit to the OHSU BDP of publicizing this unusual case of SI is described in the text, along with the number of consent forms registered with the BDP between 2018‐2019. Between 2014 and 2018, annual preregistration vacillated between 350 and 400 individuals. However, after the widespread publication of the case of SI with levocardia in 2019, the number of preregistered donors surged to approximately 460 individuals, with the majority of additional registrations taking place after publication of the findings in widely viewed media sources. The OHSU BDP undertakes other forms of outreach, though none were performed shortly before or after the SI press release in 2019.

After the poster presentation and widespread uptake of the story on media around the world, Dr. Walker was interviewed by approximately 20 media outlets, including newspapers, online news agencies, radio, and television. As a result, roughly 40 individuals who have SI with levocardia have come forward to tell their stories, with another 15 individuals who have SI with dextrocardia [right‐facing heart]. Collectively, this group, ALL of who have stated their willingness to provide medical records, be further interviewed, and participate in a future genetic study, comprises the largest group of individuals with SI who have been surveyed in the medical literature.
Comprehensive Analysis of miRNA Regulation of MGAT3 Using the miRFluR Assay
Authors: Zohora Fatema T. , Mahal Lara K.
Published: May 2022

Studies related to miRNAs and their ability to tune protein expression have gained much attention in recent years. In the field of glycobiology, miRNAs are now given special consideration as they have been shown to be key regulators of many glycosylation pathways. The enzyme MGAT3 (β‐1,4‐mannosyl‐glycoprotein 4‐beta‐N‐acetylglucosaminyltransferase) is responsible for the addition of a bisecting GlcNAc to the core mannose residue of complex or hybrid N‐glycans. This is a unique modification of complex N‐glycans that has been reported to play important roles in signal transduction, growth factor signaling, tumor progression and metastasis. In this project, we generated a comprehensive map of the regulation of MGAT3 by miRNA using a newly developed high throughput assay: miRFluR. This assay uses ratiometric analysis of a genetically encoded dual‐color fluorescence reporter (cerulean/mCherry) to identify regulatory miRNA:mRNA interactions. A dataset for the interaction between the entire human miRome (~2700 miRs) and MGAT3 has been collected and analysis of the miR regulatory map is presented. Our data provides insight into the regulation of this important glycan and its functions in disease.
Gesticulating Biochemistry: Exploring the Impact of using Augmented Reality Models on Students’ Usage of Gestures when Talking about Biochemistry
Authors: Randa Lora , Bridgeman Anna , Figueroa Vanna , Poolos Zoe , Wang Song , Bussey Thomas , Sung Rou‐Jia
Published: May 2022

There are multiple examples in the literature highlighting the connection between gestures and meaning‐making by students learning chemical concepts in chemistry; however, there is little known about whether this connection extends to student learning in biochemistry. Moreover, learning biochemistry often involves the use and coordination of multiple external representations (ranging from 2D illustrations and physical models to 3D virtual models) of macromolecules. How does use of these external representations impact student gesture production and meaning‐making when learning new biochemical content? In this study, we used a newly developed augmented reality (AR) model of the KscA channel to investigate the influence of external representations on student produced gestures during a discussion on KscA function. We focused on several research questions: what are the different biochemistry content knowledge that students express through gestures? What are the different types of gestures and patterns of gesture production used by students to express content knowledge? What are the differences, if any, in student produced gestures before and after the introduction of an AR model? What are the relationships between student produced gestures and the AR model? Semi‐structured clinical interviews were conducted and recorded through Zoom. Seven undergraduate students who enrolled in an upper‐division biochemistry course participated in the interview. During the interview, participants were presented with an AR model of KcsA channel after proposing a theory of how potassium ions move through the channel. Participants were then asked to modify their theory. In the first stage of analysis, interview participants’ utterances were transcribed and their gestures were described in terms of hand shape and movement. The identification of unique gestures as part of the coding process highlighted the importance of hand shape vs movement vs a combination of both as ways to delineate different gestures. In the second stage of analysis, qualitative and quantitative differences in student gesture production were identified through a comparative analysis of student gestures before and after their exposure to the AR model. Exposure to the AR model resulted in a shift from gestures that were more general (for example, holding two hands parallel to represent the channel) to more specific to the channel representation they say (for example, holding two hands to form a funnel shape or curling one hand into a tube shape). Our findings add to existing literature on the use of external representation in biochemistry education by highlighting the relationships between AR mediated external representations and gesture mediated external representations. Implications for both the development of new external representations and instructor’s choice of external representations are discussed.
The Role of SARS‐CoV Nonstructural Protein 1 in Cytoplasmic Stress Granule Formation
Authors: Bridges Kaitlin , Nag Anita
Published: May 2022

Severe acute respiratory syndrome coronavirus (SARS‐CoV and SARS‐CoV‐2) encoded nonstructural protein 1 (nsp1) is known to suppress host gene expression, known as host shutoff. Nsp1 interacts with host proteins that facilitate nsp1 to block host protein synthesis by cellular protein translation factors. Nsp1 also triggers the degradation of host messenger RNA (mRNA) while keeping the viral RNA intact. During viral infection, stalled host mRNAs accumulate in non‐membranous vesicles, known as stress granules (SGs). This allows mRNAs to be protected until stress ceases. Recently, our lab observed that nsp1 interacts with proteins of, and accumulates in, the SGs as well. We found SGs are smaller in size in the presence of nsp1. We aimed to determine the role of nsp1 within these granules. Given the role of nsp1 in degrading mRNAs, we suspected that this viral protein may modify the composition of SGs to prevent it from protecting host mRNAs. To identify any compositional changes by nsp1, we successfully adapted and optimized SG isolation methods in nsp1‐expressing human embryonic kidney (HEK) cells. We then further isolated the RNA following standard protocols for RNA‐seq analysis. Using immunoblot, we analyzed the proteins present in SGs isolated from cells in the presence and absence of nsp1. We were able to further isolate and identify these proteins using immunoblot and found that SG core protein, Ras‐GTPase activating protein (SH3 domain)‐binding protein 1 or G3BP1, decreases overtime in the presence of nsp1. Since nsp1 is responsible for targeting mRNA for degradation we are currently identifying mRNAs that selectively accumulate in SGs in the presence of nsp1.
Differences in Conformational Dynamics Between a Viral Polyprotein and Its Processed Products in Functionally Relevant Regions Revealed by Solution‐State NMR Spectroscopy
Authors: Winston Dennis , Boehr David
Published: May 2022

Viruses have evolved numerous strategies to expand protein function despite their limited genetic material, including proteolytic cleavage of polyproteins to obtain proteins with different functions than their precursors. In this way, proteolysis enables temporal control over the viral life cycle by varying the amounts of proteins in competing cleavage pathways. The 72 kD poliovirus polyprotein 3CD is involved in important protein‐protein, protein‐RNA and protein‐lipid interactions in viral replication and infection. It is a precursor to 3C protease and 3D RNA‐dependent RNA polymerase, but has different protease specificity, is not an active polymerase, and participates in different protein and RNA interactions than its processed products. Despite the existence of X‐ray crystal structures for the three proteins, the molecular mechanism for the different functionality of 3CD is not well understood due to the structure of the domains in 3CD being nearly identical to the isolated 3C and 3D proteins.

It has been proposed that despite nearly identical X‐ray crystal structures, the conformational dynamics of 3CD in solution may be different than those of 3C and 3D, allowing 3CD to adopt additional conformations and perform new functions compared to its processed products. To this end, we compared the conformational dynamics of 3C, 3D, and 3CD using solution‐state NMR spectroscopy. We used Carr‐Purcell‐Meiboom‐Gill (CPMG) multiple quantum relaxation dispersion, methyl 1H chemical exchange saturation transfer (CEST), and methyl spin‐spin relaxation to probe conformational dynamics from the picosecond to second timescales. We have identified differences in conformational dynamics in functionally important regions that may be useful in understanding the differences in function between 3C, 3D, and 3CD. In particular, expansion of the conformational ensemble available to 3CD may allow it to perform additional functions not seen in 3C and 3D alone despite having nearly identical lowest‐energy structures.
Mechanisms of Substrate Selectivity and Transport by a Bacterial Methionine ABC Importer
Authors: Chen Yuchun , Yang Janet G.
Published: May 2022

ABC transporters are central in many cellular functions including nutrient uptake, signal transduction membrane assembly, and cellular detoxification. Both structural and functional studies have revealed insights into the high‐affinity uptake mechanism of the MetNI methionine ABC transporter. Using the energy from ATP binding and hydrolysis, the MetNI‐Q system can import L‐Met, D‐Met, and other methionine derivatives against concentration gradients. Many mechanistic studies of ABC transporter propose a model in which cognate binding proteins sequester substrates in the periplasm and deliver them to the transporter. In contrast, recent in vivo and crystallographic studies of MetNI‐Q suggest that some substrates may be able to access the transporter through a solvent accessible tunnel in the cognate binding protein‐transporter complex. These studies suggest that the substrate delivery to MetNI transporter may be more flexible than other ABC transport systems.

To test if the MetNI‐Q system transport system uses two mechanisms of substrate delivery, we designed a series of experiments to dissect the individual steps in the transport cycle. As the starting point, we have developed a fluorescent anisotropy assay to measure the binding affinity between the MetQ substrate‐binding protein and the MetNI transporter in the presence of different methionine derivatives. Our preliminary data show that MetNI binds to apo‐MetQ ~10 fold tighter than L‐Met liganded MetQ, and additionally may differ in the presence of D‐Met. This suggests that different methionine derivatives may utilize discrete transport models.