bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti‐tuberculosis Drug Discovery from Phenotypic High‐throughput Screening of Actinomycete Cultures
Authors: Grzelak Edyta Malgorzata , Gao Wei , Choules Mary P. , Wan Baojie , Wang Yuehong , Cheng Jinhua , Lee Hanki , Suh Joo‐Won , Pauli Guido F. , Franzblau Scott G. , Jaki Birgit U. , Cho Sang‐Hyun
Published: June 2021

This report presents over a decade of efforts in anti‐tuberculosis (anti‐TB) drug discovery at the UIC Institute for Tuberculosis Research (ITR). During this period, a library of approximately 200,000 actinomycete‐derived extracts was screened directly against virulent Mycobacterium tuberculosis (M. tb) H37Rv using a luxABCDE reporter and the Microplate Alamar Blue Assay.1 The collection consisted of soil microorganisms from sites in Southeastern Asia with distinctive ecosystems and climate, e.g., polar, tropical, volcanic, and deserted areas. The extracts of interest were subjected to extensive biological profiling under replicating and non‐replicating M. tb conditions, against mono‐drug resistant M. tb strains, and panels of microorganisms other than mycobacteria. The aim was to identify and validate the extracts with the most interesting activity profile by fractionation and bioassay‐guided isolation of the active principles. New approaches were implemented not only for the biological activity testing but also for the isolation and structure identification of the potential leads.2 The established anti‐TB drug discovery platform led to the discovery of the two anti‐mycobacterial cyclic peptides, ecumicin and rufomycin, which both target ClpC1 in M. tb.

Support or Funding Information

The Next‐Generation BioGreen 21 Program (No. PJ01328403), Rural Development Administration, Republic of Korea; and T32AT007533 from NCCIH/NIH.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neuroinflammatory Responses in a Mouse Model of Tetramethylenedisulfotetramine‐Induced Status Epilepticus
Authors: Calsbeek Jonas , Gonzalez Eduardo , Boosalis Casey , Zolkowska Dorota , Rogawski Michael , Lein Pamela
Published: June 2021

Tetramethylenedisulfotetramine (TETS) is a GABAA receptor antagonist that is considered a chemical threat agent due to its ease of synthesis and its potent bioactivity in humans. Acute exposure to TETS can trigger seizures that evolve into status epilepticus (SE), and death. The current medical countermeasure for treating acute intoxication with seizurogenic chemicals like TETS includes the administration of benzodiazepines such as diazepam or midazolam. When administered 40 min post‐exposure in a mouse model of TETS‐induced SE, these positive allosteric modulators of the GABAA receptor are effective in stopping seizures and reducing mortality from ~90% to ~25%. The goal of this project was to determine the efficacy of this intervention in mitigating the neuroinflammatory response following TETS‐induced SE, with the objective of determining a feasible window of time for therapeutic intervention. In the current study, adult male NIH Swiss mice were pretreated with riluzole (10 mg/kg, i.p.) 10 min prior to receiving a lethal dose of TETS (0.2 mg/kg, i.p.). Control animals received vehicle in place of TETS and benzodiazepine. Animals were continuously monitored by EEG and/or scored for seizure behavior to confirm that TETS exposure elicited electrographic and/or behavioral seizures. At 10 or 40 min after the first myoclonic body twitch, animals were administered either diazepam (1.8 mg/kg, i.p.) or midazolam (1.8 mg/kg, i.m.) to terminate the persistent seizures. At 72h post‐intoxication, animals were perfused with fixative and brains were removed for FluoroJade C (FJC) labeling to assess neurodegeneration, and for GFAP and IBA1 immunohistochemistry to quantify neuroinflammation. Relative to vehicle control, GFAP immunoreactivity was significantly increased in the piriform cortex of all TETS‐exposed groups regardless of time of benzodiazepine administration. Midazolam was not superior to diazepam in protecting against neurodegeneration or neuroinflammation. These findings do not support the hypothesis that earlier treatment with either benzodiazepine is more effective in mitigating TETS‐induced neuropathology.

Support or Funding Information

Supported by the NINDS CounterACT Program (grant # U54 NS079202).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antiproliferative Effects of Metformin in Triple Negative MDA‐MB 231 Breast Cancer Cells Exposed to Glucose‐Starved And 2‐Deoxyglucose
Authors: Triggle Christopher , Majeed Yasser , Samuel Samson Mathews , Ding Hong Mathews
Published: June 2021

Introduction

We have previously reported that 50μM of the anti‐diabetic drug metformin protects endothelial cells against hyperglycemia‐induced endothelial dysfunction and senescence thus providing support for the vascular protective benefits of metformin when used in diabetes 1,2. However, when 2mM (but not 50μM) metformin was used in a cell culture protocol with tumor murine microvascular endothelial in a glucose‐starved (GS) or 2‐deoxyglucose (2DG; 5mM) media pro‐survival autophagy and the mTOR pathway were inhibited and cell viability was reduced 3. These data supported other published evidence from both clinical and ex vivo cell‐based studies suggesting the utility of metformin for the treatment of various cancers. We therefore postulated that triple‐negative breast cancer (TNBC) that does not benefit from hormonal or HER2 targeted therapies may also respond to a metformin and GS/2DG protocol and investigated the effects of metformin in MDA‐MB 231 TNBC cells that were cultured in the presence of 2DG.

Methods

MDA‐MB 231 cells were treated with 2DG (10mM) in a glucose free media for 48h in the absence/presence of metformin (2mM). Western blot analysis (n=3–4); cell proliferation (MTT assay; n=5); migration (Radius Cell Migration Assay; n=4) and viability (propidium iodide assay; n=5) were performed. Significance was determined by ANOVA.

Results

Cell proliferation rate significantly decreased (95%) in 1) GS + metformin when compared to non‐treated GS (88%) and 2) 2DG exposed cells treated with metformin (46%) versus cells that were treated with either metformin (20%) or 2DG (28%) alone. Cell viability significantly (P<0.05) decreased in GS treated with metformin (34%) when compared to non‐treated GS (8%). In addition cell migration in cells exposed to 2DG and treated with metformin was reduced. Western blot analysis revealed that treatment with metformin significantly decreased (fold change) the levels of LC3A‐II (2.4&1.8) and LC3B‐II (2.3&1.6), pmTOR (S2448; 1.6&1.4) and downstream p4E‐BP1(T36/47; 1.5&1.3), pS6(S235/236; 4.7&2.8) and pS6(S240/244; 3.2&1.8) in GS and 2DG‐exposed cells + 2mM metformin.

Conclusion

Results show that using mM metformin in TNBC cells in a GS media containing 2DG offers a potential adjunct anti‐cancer protocol for the treatment of TNBC.

Support or Funding Information

Supported by a Qatar Foundation National Priorities Research Program grant (NPRP: 04‐910‐3‐244) and a Junior Scientist Research Experience Program grant (JSREP: 03‐016‐3‐009)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Cannabinoid Receptor 2 Prevents Retinoblastoma Cell Migration
Authors: Vecchini‐Rodriguez Clara M. , Oquendo Yolymar , Yudowski Guillermo , Flores‐Otero Jacqueline
Published: June 2021

Retinoblastoma (RB) is a rare cancer of the retina and it is the most common intraocular malignancy present in children less than 5 years of age. Current treatments cure over 95% of RB when it is restricted to the internal part of the eye, however less than 10% of children with extraocular RB survive. The endocannabinoid system has been found to be effective anti‐cancer agents in different cancer types, yet in RB the role of cannabinoid receptors or their ligands has not been elucidated. Our main objective is to determine the role of the cannabinoid 1 receptor (CB1R) and the cannabinoid 2 receptor (CB2R) in regulating RB progression with an emphasis in tumor invasion, using human RB cells and human tumors. Data from our laboratory demonstrate that in contrast to CB1R, the RNA expression of the CB2R is upregulated in RB cells and tumors. We hypothesize that the migration and invasion of highly aggressive RB cells (Y79) is modulated specifically by the CB2R. Our results show a significant upregulation in the protein expression levels of CB2Rs in RB cells and tumors when compared to fetal retina. To elucidate the migration rate, we performed wound healing assays using Y79 cells that were exposed to a synthetic agonist that activates both, CB1Rs and CB2Rs, CP55,940. Results demonstrate a time‐dependent effect in the cell's migration upon activation of the receptors. Our data suggests a functional role for CBR2 in modulating RB cell behavior. Ongoing studies using flow cytometry assay will determine the effect of CP55,940 and 2AG, a different endogenous ligand, in promoting RB cell cycle and viability. Future experiments will test the effects of CBR2 specific ligands in halting optic nerve invasion in an in vivo orthotopic RB xenograft model, hence preventing brain metastasis.

Support or Funding Information

Grants: RCMI‐NIMHD 8G12MD007600 to JFO; NIH NIH‐NIDA R01 DA037924 to GY; NIGMS‐RISE program.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Platelets and their Pharmacological Regulation on Cancer Cell Immune Checkpoint PD‐L1 Expression
Authors: Lesyk Gabriela Maria , Poitras Erika , Jurasz Paul
Published: June 2021

Background

Cancer cells have the ability to activate platelets, and platelets play an important role facilitating hematogenous metastasis. One mechanism by which platelets promote metastasis is their ability to shield cancer cells from natural killer cell‐mediated death. Although much is known of how platelets affect the innate immune response, our understanding of platelet function in modulating the adaptive immune system in cancer is limited. A major negative regulator of the adaptive immune response in cancer is the immune checkpoint transmembrane protein Programmed Death Ligand 1 (PD‐L1). PD‐L1 interacts with its receptor PD on T‐cells resulting in T‐cell anergy and/or apoptosis. This cancer cell survival mechanism is exploited in various types of cancer, including lung and kidney. Interestingly, platelets upon activation secrete a number of factors that have the potential to increase cancer cell PD‐L1 expression. Therefore, we hypothesized that cancer cell‐activated platelets increase cancer cell PD‐L1 expression and that common anti‐platelet drugs inhibit this platelet‐induced up‐regulation of PD‐L1.

Methods

Platelets were isolated from the blood of healthy human volunteers. Human A549 lung and 786‐O renal cancer cells were incubated with and without platelets for 24 hours and cancer cell PD‐L1 surface expression was measured by flow cytometry and mRNA by qPCR. In other experiments, platelet‐cancer cell incubations were performed in the presence of anti‐platelet drugs acetylsalicylic acid (ASA – 100 mM), Prasugrel active metabolite (PAM – 10 mM) or Integrelin (10 mM).

Results

Platelets caused a significant increase in PD‐L1 surface expression by A549 (6.7±3.1% of A549 vs. 16.3±3.9% of A549 + platelets, P < 0.05) and 786‐0 cells (11.2±3.1% of 786‐O vs. 21.8±4,8% of 786‐O + platelets, P < 0.05). This increase in surface expression occurred as a result of a 3‐fold increase in PD‐L1 mRNA in A549 incubated with platelets. Platelets did not express PD‐L1 mRNA. Importantly, anti‐platelet drugs Prasugrel active metabolite (PAM) and Integrelin, but not acetylsalicylic acid, prevented the platelet‐dependent increase in PD‐L1 expression by A549.

Conclusions

Platelets up‐regulate the transcriptional expression of PD‐L1 by cancer cells, and Prasugrel and Integrelin can inhibit this up‐regulation. Further experiments are needed to determine whether platelet‐induced expression of PD‐L1 on cancer cells protects them from T‐cell induced death, and whether investigation of anti‐platelet drugs as adjuvant therapy in addition to immune checkpoint inhibitors is warranted.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of Antibiotics on Gut Microbiome: A Metagenomics Analysis of Microbial Shift and Antibiotic Resistance in Antibiotic Treated Mice
Authors: Xu Lei , Chockalingam Ashok , Stewart Sharron , Li Zhihua , Rouse Rodney
Published: June 2021

There are long standing concerns about the use of oral antibiotics and their impact on the normal gut microbiota/microbiome and the emergence of antibiotic resistance. More recently, drug‐microbiota/microbiome interactions have been identified that have impacted drug efficacy and/or safety through altered drug metabolism re‐enforcing these concerns. This project characterizes the effect of different antibiotic treatment on the composition of the gut microbiota / microbiome and the prevalence of antibacterial resistance genes in the gut of mice treated for UTI. This knowledge provides a foundation for stratifying the potential of different mono‐ and potentially combination antibiotic therapies to negatively impact health through the microbiota/microbiome. Mice were experimentally infected by inoculation of E.coli into the urinary bladder and subsequently treated with one of three antibiotics (ampicillin, ciprofloxacin, fosfomycin) commonly used for UTI. For each antibiotic, fecal samples were collected after 1, 2, or 3 days of treatment to compare and contrast influence on the gut microbiota and microbiome composition. Standard NGS procedures were used with bio‐informatics to conduct metagenome analysis on fecal samples. Although individual animal variation was observed, control animals generally had similar findings for each experiment. For each antibiotic, analysis of aligned sequences revealed a post‐treatment reduction in number of bacterial species and a shift in the relative prevalence of the remaining species. By 24 hours after treatment initiation, fosfomycin demonstrated a larger and more profound reduction in species than either ciprofloxacin or ampicillin. Within the first 24 hours, ampicillin has minimal reduction in species while ciprofloxacin was intermediate to ampicillin and fosfomycin. Ciprofloxacin and ampicillin treatment led to detection of several species not identified in controls that in some cases became one of the most prominent species detected. Fosfomycin treatment resulted in a significant increase in prevalence of a known antibiotic resistance gene in the gut microbiome. These preliminary studies indicate that the spectrum and degree of impact on the microbiota species will vary by individual antibiotic but that in all antibiotic treatments as much as 70 to 80% of detectable species are greatly reduced or eliminated. The data also show that antibiotic treatment can increase the reservoir of antibiotic resistance in the microbiome.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RNF20 regulates adipocyte differentiation through modulating PPARγ
Authors: JEON YONGGEUN , LEE JAEHO , KIM JAEBUM
Published: June 2021

During adipogenesis, numerous adipogenic transcription factors need to be sequentially expressed and tightly modulated. Among these transcription factors, peroxisome proliferator‐activated receptor gamma (PPARγ) is the most important one during adipogenesis. PPARγ is a nuclear receptor and its activity is sophisticatedly regulated through the binding of coactivators and corepressors. However, the activation mechanisms of PPARγ during adipogenesis are poorly understood. In this study, we demonstrate that the E3 ubiquitin ligase ring finger protein 20 (RNF20) would be involved in adipocyte differentiation by regulating PPARγ. In RNF20 heterozygous‐null mice, the size of adipose tissue and adipocytes were reduced. Knockdown of RNF20 suppressed adipocyte differentiation of 3T3‐L1 cells. Conversely, overexpression of RNF20 promoted adipogenesis. We observed that RNF20 bound to nuclear corepressor (NCoR1) and increased the activity of PPARγ by repressing NCoR1. Taken together, these results suggest that RNF20 would act as a positive regulator for adipocyte differentiation by enhancing the activity of PPARγ.

Support or Funding Information

This work was supported by NRF of Korea Grant MSIP 2011‐0018312 and BK21

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Method validation of analytical method for determination of extracts of rosemary in various food matrix
Authors: Choi Seung‐Hyun , Jang Gill‐Woong , Choi Sun‐Il , Jung Tae‐Dong , Cho Bong‐Yeon , Han Xionggao , Sim Wan‐Sup , Kim Young‐Cheul , Lee Ok‐Hwan
Published: June 2021

Extracts of rosemary is a food additive used as antioxidant for edible oil, dressing and sausage. In the EU and China, extracts of rosemary is authorized as food additive with maximum amounts for each food but unauthorized in Korea. Therefore, analytical method to detect extracts of rosemary in food is need. A sensitive simultaneous analytical method to identify rosmarinic acid, carnosol and carnosic acid residues in edible oil, dressing, and sausage was developed and validated using high‐performance liquid chromatography with photo diode array detector (HPLC‐PDA). Validation parameters; specifity, linearity, LOD, LOQ precision and accuracy were attained. The validated method has an analytical range from 1.56 μg/mL to 400 μg/mL in edible oil, dressing and sausage with an LOD and LOQ range from 0.22 to 1.73 μg/mL and 0.66 to 5.23 μg/mL, respectively, depending on the analytes. The recoveries of extracts of rosemary ranged from 70.6 to 116.5% with relative standard deviations between 0.2 and 3.8%. These results showed that the analytical method developed in this study in appropriate for determination of extracts of rosemary identification and can be used to maintain the safety of edible oil, dressing, sausage products containing extracts of rosemary residues.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Conformational Penalties to Assess the Structure Dependence of RNA Cellular Function
Authors: Al‐Hashimi Hashim M.
Published: June 2021

RNA is increasingly recognized as a key player in biology, a novel drug target, and a molecular tool for bioengineering applications and synthetic biology. The degree to which the cellular activity of regulatory RNAs depends on their three dimensional structure can vary from RNA to RNA and can be difficult to determine experimentally. Here, we describe a new approach for assessing the structure dependence of RNA cellular activity that relies on using mutations to introduce conformational equilibria that push the RNA toward non‐native conformations. By comparing the conformational equilibria of these mutants with their impact on cellular activity, we determine the structure‐dependence of cellular function for two highly conserved regulatory elements from HIV‐1.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein Palmitoylation Contributes to Extracellular Vesicle Production and Effect on Vascular Barrier Dysfunction during Systemic Inflammation
Authors: Yang Xiaoyuan (Sherry) , Chatterjee Victor , Creasy Alexandria , Zheng Ethan , Yuan Sarah Y.
Published: June 2021

Extracellular vesicles (EVs) are membrane‐encapsulated particles released from various types of cells, ranging from 30–1000 nm in diameter. EVs facilitate intercellular communication by transferring proteins and lipid among cells not in direct contact. Recent studies suggest the involvement of EVs in the progression of systemic inflammatory response syndrome (SIRS) associated with trauma or infection. However, there is limited information about EV production, cargo content, or pathological function in systemic inflammation. Protein palmitoylation, the covalent attachment of palmitic acid to cysteine residues, catalyzed by a family of palmitoyl acyltransferases containing a zinc finger aspartate‐histidine‐histidine‐cysteine (DHHC) motif. Palmitoylation has recently been recognized as a novel signaling mechanism in regulating endothelial barrier function and microvascular permeability. In this study, we sought to examine whether protein palmitoylation affects EV production or function in microvascular barrier in response to systemic inflammation. EVs were isolated from the blood of wild‐type (WT) and DHHC21 deficient (Zdhhc21dep/dep) mice with SIRS. Nanoparticle tracking analysis showed an increased plasma level of EVs in WT mice subjected to SIRS compared to sham‐treated, but this response was diminished in Zdhhc21dep/dep mice. Next, isolated EVs from SIRS mice were injected into healthy mice intravenously to assess their effects on microvascular permeability. Intravital microscopic imaging analysis revealed that SIRS EVs from WT mice caused a massive leakage of plasma albumin into extravascular space, whereas SIRS EVs from Zdhhc21dep/dep mice produced less leakage. Furthermore, we examined the cargo contents of EVs produced during SIRS and identified an array of pro‐inflammatory molecules, including C‐reactive protein, interleukins and tumor necrosis factor‐a. Of particular note, SIRS EVs contained a high level of histones, which has been implicated in endothelial barrier injury. Such an EV profile was not observed in EVs from Zdhhc21dep/dep mice. Taken together, these data suggest systemic inflammation stimulates the production of pro‐inflammatory EVs that are capable of inducing microvascular leakage. This response is greatly attenuated in Zdhhc21 functional deficiency, indicating the involvement of palmitoylation in EV production or function.

Support or Funding Information

This work was supported by the National Institutes of Health Grants GM097270, HL070752, and HL126646.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lysosomal Metabolic Cues in Orchestrating Cellular and Organism Homeostasis
Authors: Wang Meng
Published: June 2021

Lysosomes are highly specialized cellular organelles with crucial functions in metabolic digestion and recycling. They carry a variety of hydrolases that function at low pH and generate specific metabolic products. Work in my laboratory is interested in understanding how these metabolic activities of lysosomes are linked to their signaling communication with other cellular organelles, such as mitochondria, endoplasmic reticulum (ER) and the nucleus. We discovered a lysosome‐to‐nucleus retrograde lipid signaling pathway in promoting healthy aging and metabolic fitness, and revealed its critical roles in the metabolic and signaling interaction between lysosomes and mitochondria. Most recently, we discovered a lysosome‐specific nucleotide hydrolase and its key functions in the control of ER stress responses through interacting with mTOR signaling. These studies highlight the signaling role of lysosomes in the active coordination of cellular homeostasis and in the improvement of the healthy survival of an organism.

Support or Funding Information

This work was supported by HHMI and National Institutes of Health.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Designing new protein structures and functions with the molecular modeling program Rosetta
Authors: Kuhlman Brian
Published: June 2021

Proteins perform an amazingly diverse set of functions in all aspects of life. Critical to the function of many proteins are the highly specific three‐dimensional structures they adopt. For this reason, there is strong interest in learning how to rationally design proteins that adopt user‐defined structures. Over the last 20‐years there has been significant progress by us and others in the field of computational protein design as rotamer‐based sequence optimization protocols have been used to accurately design protein tertiary and quaternary structure. I will summarize how the molecular modeling program Rosetta is used to design new protein structures and describe how we have used this capability to create proteins that have important applications in research and medicine. I will highlight three protein design stories: (1) the engineering of light‐inducible proteins that can be used to recruit proteins to specific locations in the cell, (2) the use of protein interface design to create therapeutic bispecific antibodies, and (3) the de novo design of new protein structures from pieces of naturally occurring proteins.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lactobacillus rhamnosus GG‐mediated expression of leptin in the intestine induces cell proliferation
Authors: Darby Trevor M , Luo Liping , Naudin Crystal , Jones Rheinallt M
Published: June 2021

Identifying the functional elements that mediate efficient gut epithelial development and homeostasis is essential for understanding intestinal health and disease. Some of the molecular events that function in optimal gut epithelial health are induced after the colonization of the intestine by symbiotic bacteria. Some lactobacilli, when in contact with the gut epithelium, induce the activation of signaling pathways that function in cell proliferation, cell migration, epithelial wound sealing and cytoprotection. These processes involve the generation of non‐toxic physiological levels of ROS by NADPH oxidase (Nox1) and ensuing activation of the Nrf2 pathway signaling. However, the downstream functional elements that respond to Nox1‐generated ROS and mediate these events are yet to be fully described. Here, we show that ingestion of Lactobacillus rhamnosus GG induces elevated levels of Leptin in the gut epithelia, which as well as functioning in the context of metabolism, also has pleiotropic activity as a chemokine that induces cell proliferation. Using gut epithelial specific knockout mice, we show that L. rhamnosus GG –induced elevated levels of Leptin is dependent on a functional Nox1 in the colonic epithelium, and that L. rhamnosus GG –induced cell proliferation is dependent, on both Nox1 and on Leptin. We also show that L. rhamnosus GG induces JAK‐STAT signaling pathway in the gut in a Nox1 and Leptin dependent manner. Our data suggest that L. rhamnosus GG elevates Leptin levels in the gut epithelium, and that Leptin then acts as a chemokine promoting cell proliferation through the upregulation of JAK‐STAT pathway signaling. These results demonstrate a novel role for Leptin in the response to colonization by lactobacilli, where Leptin is involved in the transduction of signals from symbiotic bacteria to sub epithelial compartments, where it modulates intestinal development and homeostasis.

Support or Funding Information

NIH Grants R01DK098391

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Mechanisms of Cue‐Induced Translation Regulation
Authors: Filbin‐Wong Megan E. , Gonen Tamir , Kieft Jeffrey S.
Published: June 2021

Precise tuning of local gene expression is essential for proper neural development. In particular, environmental cues spatially and temporally direct neuron growth by stimulating or repressing protein synthesis. The integral membrane protein, deleted in colorectal cancer, or DCC, detects the environmental growth cue netrin‐1, and transduces the growth signal to a variety of intracellular responses, including activating protein synthesis. DCC is primed to function in this way; in the absence of netrin‐1, translation machinery is tethered to its C‐terminal tail (C‐tail) and silenced. Once netrin‐1 binds DCC, the receptor dimerizes, translation machinery is released, and protein synthesis commences. How DCC's C‐tail binds to the protein synthesis machinery and prevents translation in the absence of netrin‐1 is unknown. The C‐tail is a 35 kDa protein that is composed of three domains (P1–3) and is predicted 91% unstructured. Dimerization is facilitated by the P3 domain, while the P1 domain binds to the 60S subunit protein, ribosomal protein L5 (rpL5). Titrating the full‐length C‐tail into translation‐competent lysates, we found that the tail specifically inhibits eukaryotic translation, and inhibits protein synthesis of messages that use both canonical and non‐canonical translation initiation mechanisms. Hence, we propose that the C‐tail functions primarily through binding the ribosome, not eukaryotic initiation factors. We have evidence from cosedimentation and in vitro binding studies to show that the full‐length tail binds to the small and large subunits, as well as the 80S ribosome in the absence of mRNA. Binding and luciferase reporter assays show that the P3 dimerization domain is dispensable for translation inhibition, and the P1 domain is necessary but not sufficient to bind the ribosome and decrease translation. Thus the P2 domain and/or linker sequence(s) are also important for function. Collectively, our results suggest that DCC's C‐tail inhibits translation by effectively preventing ribosomes from binding to messages. We hypothesize the unstructured C‐tail precludes mRNA binding by interacting with the decoding groove of the ribosome until receptor dimerization releases 80S ribosomes. Thus chemoattraction via netrin‐induced DCC receptor dimerization might lead to a global (not message‐specific) increase in localized protein synthesis at axonal growth cones.

Support or Funding Information

MSU Denver Provost Seed GrantMSU Denver College of Letters, Arts and Sciences Dean's Seed Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Defining unknown steps in Coenzyme Q precursor biosynthesis
Authors: Johnson Sheila , Robinson Kyle , Pagliarini Dave
Published: June 2021

Coenzyme Q (CoQ) is a redox‐active lipid that is essential for cellular respiration and plays important roles in cellular metabolism. CoQ is produced endogenously by virtually all cells, yet multiple steps of its biosynthesis remain uncharacterized. It has been known for over fifty years that the conserved precursor for CoQ biosynthesis, 4‐hydroxybenzoic acid (4‐HB), is derived from tyrosine, but most of the metabolic steps involved in 4‐HB production have not been elucidated in eukaryotes. This pathway contains significant redundancy, and all but the first and last steps are uncharacterized. We are employing mass spectrometry, genetic screens, and classical biochemistry to discover novel intermediates and enzymes involved 4‐HB biosynthesis. Through characterization of critical enzymes and identification of pertinent intermediates, we hope to put together a comprehensive pathway detailing 4‐HB biosynthesis in eukaryotic organisms.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and Functional Characterization of the Leishmania donovani Ufm‐ylation Pathway
Authors: Lee Adrianne D , Berndsen Christopher
Published: June 2021

Ubiquitin fold modifier 1 (Ufm1) a ubiquitin‐like protein and Ubiquitin fold modifier activating enzyme (Uba5, E1) are proteins found in eukaryotic organisms that play a crucial role in cell cycle regulation, signal transduction, and ER stress. Leishmania donovani (Ld) is a trypanosomatid parasite that has been shown to have enzymes homologous to Uba5 and Ufm1. Leishmania Ufm1 and Uba5, as well as the substrate‐targeted proteins, are associated with the mitochondria which has not been observed in other organisms. This suggests that these Leishmania proteins may have physiological roles not yet described in other organisms. Leishmania donovani causes leishmaniasis, a disease that is accompanied by sores and lesions that will appear at varying depths of the body depending on the type and increases the host's susceptibility to co‐infection with other diseases. There are currently no effective vaccines for leishmaniasis. Uba5 and Ufm1 are critical for the metamorphosis and growth of the parasite. Therefore, biochemical study of these proteins may provide insight into the molecular basis for leishmaniasis and the fundamental role of this pathway in the parasite. We first expressed and purified LdUfm1, an N‐terminally truncated form of LdUfm1, and LdUba5. LdUfm1 and LdUfm1(tr.) were successfully purified and were characterized with SEC‐MALS. We then performed gel migration shift assays to demonstrate the compatibility of the human and Leishmania systems to conjugate LdUfm1. We found that full‐length Leishmania Ufm1 but not the truncated form were viable substrates for human Uba5, suggesting a role for the N‐terminus of Ufm1 conjugation. We are currently working to purify LdUba5 further for functional and structural comparisons to the human enzyme. Additionally, we began setting crystal trays to obtain protein crystals for structure determination. We have preliminarily obtained crystals of the truncated Ufm1. Future work will optimize these crystals, perform further experiments for structural characterization and explore the cross‐reaction between the human and Leishmania enzymes. Characterization of the Leishmania donovani conjugation pathway could facilitate the development of therapeutic treatments for leishmaniasis.

Support or Funding Information

4‐VA Organization,

NSF REU CHE‐1757874

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cooperativity in Proteasome Core Particle Autocatalytic Processing
Authors: Suppahia Anjana , Itagi Pushpa , Vontz Alexander , Kim Faith Mi Ge , Burris Alicia , Deeds Eric , Roelofs Jeroen
Published: June 2021

Proteasomes are complex proteases that can be found in bacteria, archaea, and eukaryotes. The formation of core particle (CP) is not trivial as it involves 28 polypeptides forming a cylindrical structure that undergoes maturation by processing of propeptides present on the β subunits. The propeptides play an important role during assembly and different roles have been described. Here, using bioinformatics approaches we identified three distinct regions in bacterial β propeptides. Molecular Dynamics simulations indicated the presence of a flexible loop‐like region (region III) that extended out of the half CP assembly intermediate for periods. We therefore hypothesized a role for region III in the dimerization of two half CPs. To test this, we used in vitro reconstitution assays with Rhodococcus erythropolis α and β subunits. Mutations in region III caused a severely reduced rate of dimerization. Region II has previously been shown to be structured and function in the association of β with two α's. When we mutated the N‐terminal region, region I, we saw an increase in rate of dimerization but a much slower rate of maturation. Since maturation requires autocatalytic removal of β‐propeptides, this suggests that the region I plays a role in efficient cleavage of the propeptide from β. The reduced maturation rate of Region I mutants allowed us to capture both pre‐holoproteasome (i.e. assembled CP with propeptides present) and holoproteasome complexes simultaneously in a reconstitution. The absence of any other intermediate forms, where a subset of the propeptides were processed, indicates that while the propeptides are processed slowly in general, the switch to holoproteasome happens within a very short time frame. This suggests the existence of cooperativity phenomenon in CP maturation. Probing for mechanisms that might explain this cooperativity, we tested for the ability of a processed β subunit to trim the propeptide of a neighboring β subunit, which we could envision as a trigger for faster processing or a conformational change. Indeed, we were able to detect cross processing of propeptides in this Rhodococcus erythropolis reconstitution assay by neighboring active β subunits both from within the same β ring as well as across the dimer interface. In conclusion, we identified novel roles of the bacterial propeptide in regulating the dimerization through region III, as well as its role in the regulating the maturation of pre‐holoproteasome through region I. Presence of two distinct populations of active and inactive CPs shows that dimerization and maturation are distinct processes.

Support or Funding Information

NIGMS R15 GM112142, R01 GM118660, K‐INBRE P20 GM103418

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evolution of Catalysis: 3′‐Phospho adenosine 5′‐phosphosulfate synthase (PAPSS) and Methionine gamma Lyase deaminase (Mgld) as an Experimental and Computational Model System
Authors: Venkatachalam KV
Published: June 2021

Binding of substrate or ligand to a protein is the first common step between a receptor protein and an enzyme. However, the receptor protein usually bear low Kd, that is higher affinity towards the ligand. Whereas, many of the enzymes bear higher Km, that is relatively lower affinity. What is the criteria behind this distinction? The product, is the transformed substrate and it would have to leave the active site so that it can perform its function outside the binding pocket or it can be transformed further by a separate set (s) enzyme(s). Between this inter play, in a bifunctional enzyme, a product from one active site pocket is a substrate for the neighboring domain. Therefore, the product of domain [(1), ATP sulfurylase] is a substrate for the domain [(2) APS kinase], the new active site. This mandates that the active site 2 must have higher affinity (low Km) than that of the domain 1, so that it stays bound tighter and gets transformed into product 2. This would avoid futile cycle of substrate of domain 2 leaving back to be bound to domain 1 to become the substrate for the domain 1 backward reaction, or to occlude/inhibit. My data, from ATP sulfurylase (domain 1) and APS kinase (domain 2) of PAPSS kinetics proves the fundamentally perfected evolutionary concept. The computational data supports this concept as well. Afterall, nature rarely fails to achieve perfection and sophistication, especially after millions of years, as part of the evolutionary process. Certain enzymes like tryptophan synthetases have developed channeling as a means to perfect its overall efficiency of ultimate product formation. PAPSS is a good model system to address the binding energies, Km, Kcat. PAPSS is a standalone enzyme that binds to five different nucleotides ATP, AMP, ADP, APS, and PAPS. In addition, this enzyme binds to two different, a phospho and a sulfo nucleotides. Methionine gammalyase deaminase (Mgld) is a different example that use PLP as a coenzyme to assist catalysis. Mgld cleaves C‐S bond of methionine to form methylthiol and the PLP bound 2‐aminobutyrate is deaminated to form alpha‐keto butyrate. Here the active site is constrained and very little flexibility is observed. Thus, the evolution of enzyme active site catalysis will be discussed using PAPSS and Mgld systems.

Support or Funding Information

N/A

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing Protein/Protein Interactions Between the Rho GTPase TCL/RhoJ and Coronin Proteins
Authors: Wilson Kaitlyn Guertin , Tader Brooke R , Hamann Michael J
Published: June 2021

TCL (RhoJ) is a Rho GTPase that contributes to tumor‐associated angiogenesis and metastatic melanoma. It is part of the Cdc42 subfamily of Rho GTPases, but it has a unique twenty amino acid N terminus that is important for GTP‐loading and localization to the plasma membrane. Deletion of the N terminus promotes GDP‐loading of TCL and its localization to intracellular vesicles. Additional data from our lab has shown that plasma membrane localization of TCL is also dependent on prenylation of the C‐terminal tail, whereas vesicular localization of TCL only required it to be in a GDP‐loaded confirmation. Thus, our hypothesis is that GDP‐loaded TCL is bound to vesicular membranes through a protein/protein interaction. Coronin proteins are logical candidates for this interaction as they bind to other Rho GTPases when they are in a GDP‐loaded conformation. To determine if coronins interact with TCL, the Cdc42‐ and Rac‐interactive binding (CRIB) domains from the seven human coronin isoforms have been PCR amplified and cloned into a pGEX plasmid for GST‐fusion protein purification. The purified CRIB will be used in precipitation assays to test for an interaction with GDP versus GTP loaded TCL. Given the importance of TCL in angiogenesis and melanoma, delineation of its interactions with partner proteins is essential for understanding its role in pathology‐related processes.

Support or Funding Information

This work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interaction between dietary vitamin A, gut microbes, and host vitamin A status
Authors: Honarbakhsh Maryam , Malta Kiana , Erricson Aaron , Zhu Chengsheng , Bromberg Yana , Zhong Guo , Isoherranen Nina , Van Buiten Charlene , Raskin Ilya , Chikindas Michael , Breslin Paul , Quadro Loredana
Published: June 2021

Micronutrient deficiency during childhood, impairs microbiome development and establishment, ultimately affecting nutrient absorption and promoting inflammatory responses, especially in the gut, in a sort of vicious cycle1. Vitamin A is an essential micronutrient that regulates many critical biological functions, such as vision, reproduction and immunity, and that more generally contributes to the integrity of the epithelia by modulating cellular proliferation and differentiation 2. Vitamin A‐deficiency (VAD) is an overwhelming and still intractable public health problem3. The essential nutrient vitamin A regulates intestinal functions by modulating inflammation, gut immunity, and intestinal cell proliferation. Thus, the vitamin A status has a profound impact on intestinal health and, indeed, VAD is often associated with pathological conditions of the gut. On the other hand, a healthy intestine is critical to support a “healthy” microbiome which in turn helps maintaining proper intestinal functions in a feedback loop.

To gain further understanding of the complex interplay between vitamin A, host physiology and microbiome, we used Lrat−/−Rbp−/−mice as a model of marginal VAD 4, when maintained on a vitamin A‐sufficient diet, and of severe VAD when deprived of dietary vitamin A. Due to the absence of lecithin:retinol acyltransferase (LRAT) and retinol‐binding protein (RBP), these mice cannot store and mobilize, respectively, hepatic vitamin A towards the periphery of the body. Thus, they rely exclusively on dietary vitamin A to support vitamin A‐dependent functions4. At 6 weeks of age, Lrat−/−Rbp−/− and WT mice were placed on either vitamin A‐sufficient or ‐deficient diet for 4 weeks and then returned to the initial regular chow regimen for 1 week. Various tissues, including intestine, and feces were collected at different time points throughout the experiment. We confirmed that VAD impairs intestinal vitamin A homeostasis and barrier functions (reduced antimicrobial defense and mucus production; increased oxidative stress; leaky gut). Genomic sequencing of the fecal 16S RNA showed that fecal dysbiosis is concomitant with the VAD status rather than the dietary vitamin A content per se, even though the degree of deficiency (marginal vs severe) did not further affect the dysbiosis. Instead, a preliminary analysis of microbial functionalities showed a significant separation of the microbial populations between vitamin A sufficient, marginal VAD and severe VAD.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization of UBE2O as a New β‐arrestin Interactor
Authors: Sokrat Badr , Bouvier Michel
Published: June 2021

β‐arrestin plays a major role in GPCR regulation and can act as a scaffolding protein for activation of non‐canonical signaling pathways. Thus, it is important to improve our knowledge of β‐arrestin interactors and the role of these protein complexes in GPCR regulation and signaling. The goal of this project was to identify new β‐arrestin interaction partners using the BioID proteomic approach. The BioID technique consists in the fusion of the biotin ligase protein BirA to β‐arrestin and exploits BirA capacity to biotinylate proteins in its proximity. The biotinylated proteins can then be isolated using streptavidin beads and identified by mass spectrometry.

We identified 37 potential interactors for β‐arrestin 1 and 86 potential interactors for β‐arrestin 2 localized in different cellular compartments and involved in many different cellular functions and signaling pathways. One of the potential interactors identified is the atypical ubiquitin ligase UBE2O, which caught our attention due to the importance of ubiquitination in β‐arrestin and GPCR regulation. We confirmed the interaction between β‐arrestin 2 and UBE2O by co‐immunoprecipitation. UBE2O is known for its role in the monoubiquitination of BAP1, a tumor suppressor protein that interacts with chromatin‐associated proteins and regulates cell proliferation, resulting in its cytoplasmic sequestration. However, UBE2O function in β‐arrestin and GPCR signaling remains unknown. Considering that β‐arrestin shuttles between the cytoplasm and nucleus, UBE2O could play a role in the regulation of its cellular localization. Using different proteomic and biochemical approaches, we explored the function of UBE2O in GPCR and β‐arrestin ubiquitination and its potential effects on GPCR signaling and trafficking. We were able to identify by mass spectrometry UBE2O substrates involved in GPCR signaling and we showed that UBE2O affects β‐arrestin and GPCR trafficking by ebBRET (Enhanced bystander bioluminescence resonance energy transfer). Considering GPCRs physiological and therapeutic importance, this project will allow improvement of our understanding and knowledge on ubiquitination regulation of GPCR signaling and could contribute to revealing new signaling modalities that might have therapeutic relevance.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.