bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 Information4‐VA Organization,NSF REU CHE‐1757874This 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 InformationNIGMS R15 GM112142, R01 GM118660, K‐INBRE P20 GM103418This 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 InformationN/AThis 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 InformationThis 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.
Uric Acid Activates Aldose Reductase and the Polyol Pathway for Endogenous Fructose Production and Fat Accumulation in the Development of Fatty Liver
Authors: Lanaspa Miguel A. , Andres‐Hernando Ana , Johnson Richard , Sanchez‐Lozada Laura G 
Published: June 2021

Dietary sugars containing fructose have been strongly associated with the development of fatty liver, but recent studies also suggest fructose can be produced in the liver via the polyol pathway where it may also induce hepatic fat accumulation. Here we investigate whether uric acid regulates aldose reductase, a key enzyme in the polyol pathway. We evaluated whether soluble uric acid regulates aldose reductase expression both in cultured hepatocytes (HepG2 cells) and in the liver of hyperuricemic rats, and whether this stimulation was associated with endogenous fructose production and fat accumulation. Uric acid dose stimulated the expression of aldose reductase in HepG2 cells in association with endogenous fructose production and triglyceride accumulation. The mechanism was mediated by uric acid‐induced oxidative stress with the stimulation of the transcription factor NFAT5. Uric acid could amplify the effects of elevated glucose levels to stimulate hepatocyte triglyceride accumulation. Hyperuricemic rats also showed elevated hepatic aldose reductase expression, endogenous fructose accumulation and fat accumulation that was significantly reduced by co‐administration of the xanthine oxidase inhibitor, allopurinol. These studies suggest that uric acid, which is generated during fructose metabolism, may act as a positive feedback to stimulate endogenous fructose production by stimulating aldose reductase in the polyol pathway. These studies provide an amplifying mechanism by which soft drinks rich in glucose and fructose can induce non‐alcoholic fatty liver disease.Support or Funding InformationSupported by NIH Grant R01 DK108859This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered Small Intestinal Morphology and Whole‐Body Energy Homeostasis in the Intestinal Fatty Acid Binding Protein (IFABP) Knockout Mouse
Authors: Lackey Atreju I , Chen Tina , Zhou Yin X , Bottasso Arias Natalia M , Gajda Angela M , Joseph Laurie B , Storch Judith 
Published: June 2021

Intestinal‐fatty acid binding protein (IFABP; FABP2) is a 15 kDa intracellular protein present in high abundance in the cytosol of the small intestinal (SI) enterocyte. High fat (HF) feeding of IFABP−/− mice resulted in lower weight gain and reduced fat mass relative to WT mice, revealing that ablation of this intestine‐specific protein leads to dramatic whole‐body phenotypic alterations (Gajda et al., JBC 2013 288:30330–30344). To understand the mechanisms leading to these differences in systemic energy homeostasis, we examined intestinal properties that may underlie the observed lean phenotype. Fecal lipid analysis demonstrated that IFABP−/− mice did not differ in their fecal lipid content, suggesting they are not malabsorbing dietary fat. However, the total excreted fecal mass for the IFABP−/− mice was significantly increased relative to WT mice. Moreover we found reduced intestinal transit time in the IFABP−/− mice, which underlies the increased fecal excretion. During tissue collection it was noted that the small intestine of IFABP−/− mice appeared to be more fragile, thus we questioned whether they may have alterations in intestinal structure. Histological analysis demonstrated that IFABP−/− mice have a shortened average villus length, reduced goblet cell density, and a reduced Paneth cell abundance relative to WT mice. BrdU proliferation assays demonstrated that the amount of proliferating cells in the crypts of IFABP−/− mice does not differ from that of WT mice, suggesting that the blunt villi phenotype is likely not due to alterations in proliferation. IHC analyses revealed increased staining for markers of inflammation, which is often observed in animals that have increased intestinal permeability. Taken together, while the effects of IFABP ablation within the enterocyte may be relatively modest in terms of lipid, there are clear changes in gut motility and morphology which likely contribute to the dramatically different phenotypes occurring at the whole‐body level. Thus, these results suggest that IFABP is likely involved in dietary lipid sensing and signaling, influencing intestinal motility, nutrient absorption, and intestinal structure, thereby impacting systemic energy metabolism.Support or Funding InformationThis work was supported by National Institutes of Health Grant DK‐38389 and NJAES (to J. S.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alterations in Nuclesome Core Particle Dynamics in the Context of Paraganglioma
Authors: Amato Matthew , Maher Louis Jim , Becker Nicole , Botuyan Maria Victoria , Zhao Debiao Jim 
Published: June 2021

Paraganglioma (PGL) is a rare form of neuroendocrine cancer for which the underlying molecular basis is poorly understood. A mysterious hallmark of many PGL tumors is the inactivation of succinate dehydrogenase (SDH), a mitochondrial enzyme of the TCA cycle. Why does SDH loss induce cancer in certain cells? As a result of SDH loss, PGL tumor cells accumulate succinate, which may have epigenetic consequences. For example, certain lysine residues on histone proteins in nucleosomes are subject to succinylation by succinyl‐CoA as a post translational modification (PTM) that converts the lysine cation to an anion. We hypothesize that the structure and stability of the nucleosome core particle (NCP) is affected by succinylation PTMs through weakening of electrostatic histone‐DNA and histone‐histone interactions. Proteomic analysis of an SDH‐loss cell line model of PGL previously demonstrated that the greatest increase in nucleosomal lysine succinylation maps to H4K79 when compared to a control cell line. The mammalian H4K79 residue lies within the nucleosome core near wrapped DNA. We specifically hypothesize that the charge inversion conferred by H4K79 succinylation, destabilizes the NCP. Point mutations at H4K79 have been generated as lysine succinylation mimics at this site. Biophysical and in vitro transcription assays are being utilized to validate the assembly of NCPs incorporating lysine succinylation mimics at this site and to interrogate their resulting stabilities. Analyses of alterations in NCP dynamics in variant NCP will help to elucidate the role of lysine succinylation of histones in the altered chromatin landscape observed during PGL oncogenesis.Support or Funding InformationGM75148‐09‐10 PREPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.