bluuebunny/crossref_metadata_2025 huggingface dataset:
Time Resolved IRMS Study of Staurosporine Induced Apoptosis in Murine Astrocyte Cells
Authors: Weber Kayla N , Husher Sara C , Mazzer Paula A
Published: November 2023

The advent of genomic and post genomic technologies is transforming the field of toxicology, and is moving the discipline from the realm of classical biology into those of molecular biology and bioanalytical chemistry. At the molecular level, it is possible to focus on both the direct impact of toxic insult as well as the cellular or organismic response to the resulting damage. The “‐omics” technologies—genomics, proteomics, and metabolomics—are likely to lead the way in the development of approaches to molecular pathology. Metabolomics, in fact, shows such promise in understanding the biochemical functioning of cells that the development of metabolomic technologies has been a major NIH roadmap initiative. One subspecialty of metabolomics, lipidomics, shows a special promise because lipids are simple to analyze and have short response time to toxic insult. One shortcoming of current lipidomic exploration is a lack of knowledge of which lipid synthetic pathways are activated in response to toxic insult. Therefore, we undertook to study lipid synthesis in response to apoptotic cell death in murine (C8‐D1A ATCC) astrocyte cells.

To investigate time resolved lipid expression we used a model of staurosporine‐induced apoptosis in murine astrocyte cells. We extracted lipids at defined time points after staurosporine insult using a modified Bligh and Dyer technique. Lipids were then sorted by lipid class using rapid HPTLC and then derivatized to fatty acid methyl esters (FAMEs). Time dependent changes in fatty acid expression were observed over the first six hours following staurosporine treatment. Inclusion of 13C‐labeled glucose immediately prior to staurosporine addition allowed for investigation of de novo fatty acid synthesis by Isotope Ratio Mass Spectroscopy (IRMS). The ability to use IRMS to trace formation of lipid synthesis has the potential to add a new dimension to future lipidomic studies.

Support or Funding Information

This work was sponsored by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103443.
Readers, writers and erasers of nuclear PIP3.
Authors: Blind Raymond D
Published: November 2023

Structural analyses of the NR5A nuclear receptors (NR5A1 and NR5A2) show that PIP2 and PIP3 bind these transcription factors in a format where the acyl chains are buried deep in the core of the NR5A protein, while the phospholipid headgroups are solvent exposed. We previously demonstrated that the phosphoinositide headgroups of these complexes are accessible to nuclear lipid signaling enzymes, which directly modify these transcription factor‐bound PIPs, altering the transcriptional output from NR5As. Here, we took a proteomics approach to ask if the solvent exposed PIP3 headgroup is available to interact with nuclear proteins. We identified two PH‐domain containing proteins that interact with NR5A1/PIP3, but not other phospholipid bound forms of NR5A1, and siRNA knockdown of these two genes decreases NR5A1 transcriptional activity. PH‐domains are one of the most well represented domain classes in the human genome, and are widely thought to mediate membrane binding, however only 10% of the PH‐domain proteins in yeast bind membranes. Our data suggest a fraction of PH‐domain containing proteins act as readers of nuclear PIP3, while nuclear lipid signaling enzymes acts as writers and erasers of nuclear PIP3. These data provide a direct mechanism that explains how nuclear PIP3 mediates transcriptional activation in mammalian cells.

Support or Funding Information

Jimmy V Foundation V‐Scholar Young Investigator Award, Vanderbilt Diabetes Research and Training Center Pilot & Feasibility Award on NIDDK P30 DK020593, Vanderbilt Diabetes Center Discovery Award, American Cancer Society/Vanderbilt‐Ingram Cancer Center Institutional Research Grant IRG‐58‐009‐56.
Differential growth state‐dependent activation of p38MAPK by treatment of endothelial cells with docosahexaenoic acid (DHA)
Authors: Du Youjia , Taylor Carla G , Aukema Harold M , Zahradka Peter
Published: November 2023

Introduction

Endothelial cells line the luminal surface of blood vessels and play an essential role in regulating vascular function. Endothelial cells quiescence is a primary property of the healthy endothelium, whereas cell proliferation is feature of vessel repair and progression of atherosclerosis. Docosahexaenoic acid (DHA), an omega‐3 fatty acid present in fish oil, is thought to exert an array of beneficial effects on vascular health by modulating endothelial function. However, a comparison of the effects of DHA on growing and confluent endothelial cells, which represent repairing and healthy states, respectively, has never been reported.

Objective

To investigate the effects of DHA on viability and proliferation of subconfluent (growing) and confluent (quiescent) human endothelial cells (EA.hy926) in relation to p38 mitogen‐activated protein kinase (MAPK) activation.

Methods

EA.hy926 endothelial cells were seeded on Matrigel‐coated plates and grown up to 10 days. Cells were treated with DHA (1–150 μM) for 24 h when they were either subconfluent (4 d after seeding) or confluent (8 d after seeding). Viability and proliferation were assessed. Phosphorylation of p38MAPK and mitogen‐activated protein kinase kinase (MKK3/MKK6) was assessed after treating subconfluent and confluent cells with 125 μM DHA over 8 h.

Results

Maximum cell number and DNA synthesis rate were reached on day 8, and the cells subsequently became quiescent. DHA >125 μM reduced viability of both growing and confluent cells after 24 h, while 40 μM slowed proliferation of both subconfluent and confluent cells. In subconfluent cells, 125 μM DHA activated p38MAPK and MKK3/MKK6 (a known upstream kinase), producing peaks at both 10 min and 4 h. In contrast, phospho‐p38MAPK and phospho‐MKK3/MKK6 peaks were only observed at 10 min in confluent cells.

Conclusions

Our study is the first to show a dose‐dependent effect of DHA on both proliferating and confluent endothelial cells with regards to viability and proliferation, as well as a growth state‐dependent activation of p38MAPK. This is highly relevant to how blood vessels respond to a dietary constituent such as DHA in various disease states. Since activation of stress‐stimulated protein p38MAPK, which is closely linked to pathological vascular remodeling, was observed in different growth conditions of cells, further studies to delineate the physiological effects of DHA on p38MAPK signal transduction in endothelial cells are warranted.
Tocopherols and tocotrienols prevent lipoxygenase‐driven phospholipid oxidation in ferroptosis
Authors: Mao Gaowei , Qu Feng , Angeli Jose Pedro , St Croix Claudette , Dar Haider , Tyurin Vladimir , Ritov Vladimir , Kapralov Alexandr , Amoscato Andrew , Anthonymuthu Tamil , Mohammadyani Dariush , Yang Qin , Stockwell Brent , Tyurina Yulia , Conrad Marcus , Bayır Hülya , Kagan Valerian
Published: November 2023

Ferroptosis, a recently discovered regulated necrotic pathway, has been claimed to include a required stage of lipid peroxidation. However, neither the nature of oxidized lipid species nor their subcellular location and enzymatic generators have been identified. We established that inactivation or depletion of glutathione peroxidase 4 (Gpx4) induced ferroptosis in mouse embryonic fibroblasts (MEFs). Using mitochondria and endoplasmic reticulum (ER) targeted fluorogen‐activating proteins (Mito‐FAP and ER‐FAP), we demonstrated that generation of oxidized lipids predominantly occurred in the ER compartment. We further showed that oxidized arachidonoyl‐ and adrenoyl‐phosphatidylethanol‐amines (but not oxygenated free fatty acids) were the major players in the execution of ferroptosis. Formation of the oxidized phospholipids was predominantly catalyzed by 15‐lipoxygenases (LOX). Vitamin E homologues (tocopherols and tocotrienols) bind with the catalytic site of LOX and suppress ferroptosis, revealing a potential non‐canonical mechanism of action of vitamin E in protection against ferroptosis likely realized independently of radical scavenging. Taken together, these findings decipher molecular mechanisms of ferroptosis and provide new therapeutic targets.

Support or Funding Information

US National Institutes of Health (NS076511 to V.E.K., NS061817 to H.B., and ES020693 to Y.Y.T.), the Human Frontier Science Program (HFSP‐RGP0013/2014), and the Deutsche Forschungsgemeinschaft (CO 291/2‐3 and CO 291/5‐1) to M.C.
Differential effects of lipids on cellular activities of SH2 domain‐containing Proteins
Authors: Singaram Indira , Cho Wonhwa
Published: November 2023

SH2 (Src Homology 2) domain is a prototypical protein interaction domain that specifically recognizes a phosphorylated tyrosine (pY) motif. Our recent study showed that a large majority of human SH2 domains bind lipids in the plasma membrane (PM) with high affinity using an alternate cationic patch (ACP) that is topologically distinct from the cationic pY‐binding pocket. However, some SH2 domains lacking ACP bind PM lipids, including phosphoinositides, in their pY‐binding pockets. To understand the differential effects of lipids on ACPs and pY pockets of SH2 domains on the cellular activities of their host proteins, we performed computational, biophysical and cellular activity and imaging analyses with various SH2 domain‐containing proteins and their lipid‐binding site mutants. Our results demonstrate highly variable mechanisms by which lipids cellular protein‐protein interactions and modulate signaling activities of these proteins in immune cells.

Support or Funding Information

NIH GM68849
Targetting Cysteinyl Leukotriene 2 Receptor as a Therapeutic Target for Tumor Growth and Metastasis: A New Function for an Old Receptor
Authors: Teegala Lakshminarayan Reddy , Duah Ernest , Kondeti Vinay , Al‐Azzam Nosayba Zakariya , Adapala Ravi , Ghebreigziabher Samrawit , Thodeti Charles , Paruchuri Sailaja
Published: November 2023

Excessive or insufficient angiogenesis can lead to number of diseases including atherosclerosis, diabetic retinopathy, age‐related macular degeneration and cancer. Although long‐standing inflammation secondary to chronic infection or irritation mediated by immune cells has been implicated in cancer progression, the contribution of endothelial cells (EC) in the generation of inflammatory mediators and their effects on angiogenesis is relatively unknown. One of the pro‐inflammatory mediators produced by EC are cysteinyl leukotrienes (Cys‐LTs: LTC4, LTD4 and LTE4), and they mediate their effects through two main receptors, CysLT1R and CysLT2R. Cys‐LTs have been shown to be involved in several human cancers including breast cancer and melanoma. Tumor microenvironment comprises numerous signaling molecules and pathways that influence angiogenic response leading to increased and aberrant vascularization. Further, angiogenesis is required for the supply of nutrients and oxygen for tumor growth and also serves as a route of tumor cells metastasis. Therefore, we investigated the role of CysLTR in angiogenesis, tumor progression and metastasis, using mice lacking CysLT1R (CysLT1RKO) and CysLT2R (CysLT2RKO).

We observed enhanced CysLT2R expression, and not CysLT1R in wild type (WT) tumors and tumor growth as well as angiogenesis were significantly decreased in CysLT2RKO mice compared to WT and CysLT1RKO mice in Lewis lung carcinoma (LLC) tumor model. Further, though few in number, tumor vessels in CysLT2RKO mice showed intact pericyte coverage and reduced permeability with simultaneous reduction in tumor cell metastasis to the lung. Furthermore, we found that activation of CysLT2R, but not CysLT1R increased EC contraction leading to junctional destabilization and permeability in vitro. Importantly, our results show that CysLT2R activates Rho kinase and inhibition of Rho kinase significantly attenuated CysLT2R‐induced EC contraction and permeability. Further, our preliminary results revealed that pericytes express CysLT2R and display calcium influx in response to LTD4. Taken together, our results suggest that CysLT2R promotes leaky blood vessel formation, enhancing tumor growth and metastasis to the lung. Blocking CysLT2R could offer novel CysLT2R‐targeted, therapeutic candidates for the treatment of cancer and other angiogenic disorders.

Support or Funding Information

James Foght Professorship Support, AHA GIA 15GRNT 25670004
Sac1 degrades its lipid substrate PI4P in the ER to maintain a steep electrochemical gradient on donor membranes
Authors: Hammond Gerry , Wills Rachel , Zewe James
Published: November 2023

PI4P metabolism has been proposed to form the basis of an “inositide motive force”, whereby a steep electrochemical gradient of this lipid that is high in a target membrane and low in the endoplasmic reticulum (ER) drives counter transport of other lipids. This mechanism assumes that the ER‐resident Sac1 phosphatase degrades PI4P in the ER (i.e. acting in cis). However, several studies have proposed that the enzyme instead operates in trans at contact sites between the ER and other PI4P rich membranes. We therefore sought to establish whether Sac1 truly acts in cis or trans in living cells. Compared with bona fide plasma membrane (PM)‐ER contact site proteins and broadly distributed ER proteins, Sac1 exhibits a localization consistent with the latter. Strategies to test activity of the enzyme at the PM in living cells revealed far more robust activity when the enzyme met its substrate in cis than at contact sites in trans. Finally, we used chemical and genetic strategies to inactivate Sac1 in yeast and mammalian cells, revealing dramatic accumulation of PI4P in the ER. This result is consistent with a failure to degrade the lipid only after its delivery to the ER. Together, the data show that Sac1 acts in cis, establishing the plausibility of an inositide motive force, and implicating PI4P transfer proteins as essential to phosphoinositide and lipid homeostasis.

Support or Funding Information

1R35GM119412‐01: NIH(NIGMS) R35
Cholesterol Hydroperoxide Trafficking: Impairment of Macrophage Cholesterol Efflux with Implications for Atherogenesis under Oxidative Stress
Authors: Korytowski Witold , Wawak Katarzyna , Pabisz Pawel , Chadwick A C , Sahoo D , Girotti Albert W
Published: November 2023

This study describes a type of “stealthy” delivery of cholesterol 7α/β hydroperoxides (7‐OOH) via a natural cholesterol trafficking pathway. In vascular macrophages this pathway normally delivers cholesterol to mitochondria to initiate export signaling when its level becomes excessive. Because 7‐OOHs are structurally similar to cholesterol, they can be recognized by steroidogenic acute regulatory (StAR) proteins and trafficked to mitochondria alongside cholesterol.

Oxidative stress associated with cardiovascular disease can produce various oxidized lipids, including cholesterol oxides such as 7‐OOH, 7‐hydroxide (7‐OH), and 7‐ketone (7=O). Unlike 7=O and 7‐OH, 7‐OOH is redox‐active, giving rise to the others via potentially toxic free radical reactions. In the present study we tested the novel hypothesis that under oxidative stress conditions, StAR family proteins not only deliver cholesterol to/into mitochondria of vascular macrophages, but also 7‐OOH, which induces peroxidative damage that impairs early stage reverse cholesterol transport. Stimulation of human monocyte‐derived THP‐1 macrophages with dibutyryl‐cAMP resulted in substantial upregulation of StarD1 and plasma membrane ABCA1 which exports cholesterol to acceptors such as ApoA1. siRNA‐induced StarD1 knockdown (kd) prior to stimulation had no effect on StarD4, but reduced ABCA1 upregulation, linking the latter to StarD1 functionality. Mitochondria in stimulated StarD1‐kd cells internalized 7‐OOH slower than non‐stimulated controls and underwent less 7‐OOH‐induced lipid peroxidation and membrane depolarization, as probed with C11‐BODIPY and JC‐1, respectively. Major functional consequences of 7‐OOH exposure were (i) loss of mitochondrial CYP27A1 activity, (ii) reduced 27‐hydroxycholesterol (27‐OH) output, and (iii) down‐regulation of ABCA1 and ABCG1. Consistently, 7‐OOH‐challenged macrophages exported less cholesterol to apoA‐I or HDL than did non‐challenged controls. StarD1‐mediated 7‐OOH transport was also found to be highly cytotoxic, whereas 7=O and 7‐OH were minimally toxic. These findings support our hypothesis that 7‐OOH found in oxidized low density lipoprotein gets delivered to macrophage mitochondria and promotes atherogenesis through impairment of cholesterol homeostasis.

Support or Funding Information

This work was supported by Polish National Science Center grant 2014/13/B/NZ3/00833 (WK) and by National Institutes of Health grants HL85677 (AWG) and HL58012 (DS). Support of American Heart Association Fellowship 14PRE185800221 (ACC) is also acknowledged
Functional Reconstitution of the Beta Cell Porosome
Authors: Naik Akshata R. , Lewis Kenneth T , Jena Bhanu P.
Published: November 2023

Supramolecular cup‐shaped lipoprotein structures called porosomes embedded in the cell plasma membrane mediate fractional release of intravesicular contents from cells during secretion. The presence of porosomes, have been documented in many cell types including neurons, acinar cells of the exocrine pancreas, growth hormone secreting cells of the pituitary, and insulin‐secreting pancreatic beta cells. Mouse insulin‐secreting Min6 cells possess ~100 nm in size porosome complexes, composed of nearly 30 proteins. Isolated Min6 porosomes reconstituted into live Min6 cells demonstrate elevated levels of porosome proteins and an increase in the potency and efficacy of glucose‐stimulated insulin release. Increased glucose‐stimulated insulin secretion up to two days following porosome reconstitution, reflects on the stability and viability of reconstituted porosomes in live cells. These results, establish a new paradigm in porosome‐mediated insulin secretion in beta cells.
A Membrane Trafficking Screen To Identify Components Involved In Clathrin‐Independent Endocytosis
Authors: Dutta Dipannita , Wayt Jessica , Donaldson Julie G
Published: November 2023

Endocytosis is an important event that cells utilize to internalize cell surface proteins and fluid into the cell. There are two main forms of endocytosis: clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE), amongst which CME is most studied. While CME is important for the internalization of many surface receptors, bulk trafficking of membrane and fluid occurs primarily in a clathrin‐independent manner. Our lab has focused on CIE associated with Arf6, although other CIE pathways exist for specific cargoes and in different cell types. While we have a fair understanding of the trafficking and sorting of the cargoes that utilize CIE, we lack understanding of the entry mechanism. We know that CIE occurs independently of clathrin, the clathrin adaptor protein (AP2), and dynamin. Among the types of CIE cargo proteins, we have focused on two ubiquitously expressed proteins, the major histocompatibility complex class I (MHCI) and CD59, a GPI‐anchored protein, with an objective to identify requirements for their entry. Therefore, we performed an siRNA screen using the Dharmacon™ Membrane Trafficking library, which targets 140 genes, to identify proteins essential for the internalization of CD59 and MHCI in Hela cells. Target genes were individually knocked down and cells were allowed to internalize CIE cargo proteins. Following internalization, the proteins were visualized by immunofluorescence and the amount of internalized protein was quantified by image analysis software. Depletion of each candidate gene was scored as either inhibiting or enhancing internalization based on the relative amount of internalization compared to that observed with the non‐target siRNA control cells. The knocked‐down genes that lead to inhibition of internalization might represent components involved in CIE entry and the knocked‐down genes that lead to “enhanced” internalization could be negative regulators of this process. The hits from the initial screen were followed up by other confirmatory tests with individual siRNA oligo sequences. Interestingly, different proteins appeared as hits for MHCI and CD59 suggesting the differential regulation of integral membrane proteins vs. GPI‐anchored proteins in CIE. Surprisingly, among them are distinct subunits of AP2 that inhibited MHCI internalization specifically, but not CD59. Knocking down individual alpha or beta subunits of AP2 inhibited MHCI internalization whereas knockdown of the mu subunit, which is typically knocked down to demonstrate a requirement for AP2/clathrin, had no such effect. This raises the question of whether components of AP2 can function in CIE entry independent of the involvement of CME. We hope to understand the function of these two AP2 subunits in CIE in future studies.

Support or Funding Information

National Institutes of Health
Palmitoylation Impact on the Sodium Hydrogen Exchanger Isoform 1 Function
Authors: Pritsch Emily , Holland Alexander , Kooiker Amanda J , Rastedt Danielle E , Vaughan Roxanne A , Foster James D , Wallert Mark Anthony , Provost Joseph John
Published: November 2023

The sodium hydrogen exchanger isoform 1 (NHE1) regulates intracellular pH and directs protein motility as an anchor for proteins at the leading edge of migrating cells. NHE1 has three putative palmitoylation cysteine targets in its cytoplasmic tail. Palmitoylation is a reversible, post‐translational fatty acid modification (S‐acetylation) of cysteine residues altering cellular location and function of many proteins. 2‐bromopalmitate (2BP) is a non‐metabolizable palmitate analog that irreversibly inhibits the protein acyltransferases responsible for palmitoylation. Using a live‐dead assay we found that up to 50 μM 2BP had a minimal (8.5 −/+ 2.5 % SEM) effect on cell viability and was not significantly different than the control palmitate at all concentrations. A 25% reduction in NHE1 palmitoylation was observed after the addition of 15 μM 2BP over 18 hours of incubation. Incubation of 15 μM 2BP in lung fibroblasts expressing human NHE1 (PSN) abrogated cell proliferation in low serum (0.5% FBS) conditions and reduced proliferation four fold in the presence of 10% FBS. Interestingly cells incubated with 2BP show a diminished adhesive ability on quartz coverslips. LPA induced stress fiber formation was decreased in cells treated with 2BP compared to vehicle control. Transiently transfected RFP tagged NHE1 cellular location and migration was determined in the absence and presence 2BP. Finally, we show the impact of 2BP and its control, palmitate, on intracellular pH indicating a role of palmitoylation on NHE1 function. Together this work shows a novel and yet undiscovered regulation of an important protein involved in intracellular pH homeostasis and directed cell motility.

Support or Funding Information

This work was funded in part by the Beckman Scholars Program, National Institutes of Health grants DA 031991 (JDF), DA13147 and 5P20‐104360 (RAV), P30‐GM103329 (UND), P20‐GM12345 (UND)
Mutation of Follicle Ftimulating Hormone Receptor Putative Caveolin Binding Motif Results in Altered Signaling
Authors: Fleischer Justin H. , Cohen Brian D.
Published: November 2023

The human follicle stimulating hormone receptor (hFSHR) is a glycoprotein hormone receptor belonging to the g protein‐coupled receptor family. It is important in both male and female reproductive processes; defects in hFSHR can lead to infertility, delayed puberty, reduced muscle bulk, and osteoporosis. Work in other labs has shown that GPCRs can be localized to microdomains located within the cell membrane called lipid rafts. These regions are highly resistant to detergents because of the high concentration of sphingolipids and cholesterol. Also within these domains, an intracellular protein, caveolin, is present. Our lab has shown that hFSHR also localizes to lipid raft domains. We hypothesize that interaction with caveolin is important in the raft residency of hFSHR through a putative caveolin binding motif (CBM). The interaction between caveolin and hFSHR in these microdomains may regulate intracellular pathways activated by the receptor.

To test our hypothesis, we have constructed CBM mutants and are creating stable cell lines expressing the mutant receptors to study the effects of mutating the key phenylalanine residues of the CBM. Interestingly, wild type FSHR presents as a doublet on a western blot while the mutants only have a single band. We theorize that one of these bands may be a glycosylation variant however further investigation is required. The mutated receptors qualitatively show normal p44‐MAPK signaling in response to FSH, indicating that they are reaching the cell surface and are capable of binding hormone and coupling to signaling partners. We have observed a subtle difference in downstream PKA targets between the mutant and wild type hFSHR, suggesting that there might be a difference in the ability to activate downstream targets. Studying the interaction between hFSHR and caveolin has the potential to develop new contraceptive antagonists and alternative agonists to regulate fertility.
Lipid Raft Disruption Alters Human Follicle Stimulating Hormone Receptor Signaling in a Human Granulosa Cell Line
Authors: Pradhuman Jenna G. , Geisel Gregory R. , Cohen Brian D.
Published: November 2023

Human follicle stimulating hormone (hFSH) is a gonadotropin responsible for stimulating gonads and is necessary for regulating reproductive systems in both females and males. The hFSH receptor (hFSHR) is a seven transmembrane receptor that belongs to the G protein coupled receptor family. The receptor is attached to a G protein on the inside of the cell. Once FSH activates its receptor, the G protein is then activated and starts a cascade of signaling that results in activation of protein kinase A (PKA) and the p44/42 MAP kinase (MAPK). The activation of these secondary proteins is responsible for follicular stimulation and gametogenesis. Previous work in the Cohen Lab has shown that hFSHR is located in cholesterol and sphingolipid enriched microdomains referred to as lipid rafts. These microdomains are detergent resistant membranes and float on sucrose gradients. In an HEK293 cell line stably expressing hFSHR, disruption of lipid rafts by the cholesterol chelator methyl beta‐cyclodextrin (MβCD) interferes with PKA activation. Current research is focused on the relevance of hFSHR lipid raft residency in the human granulosa cell line hGrC1; focusing in particular on the activation of signal transduction pathways by hFSHR. Surprisingly, disruption of the lipid rafts by MβCD in hGrC1 cells resulted in increased signaling from hFSHR. However, it is unknown if the signaling change is because of cholesterol depletion or lipid raft disruption. Further studies will attempt to parse out this difference. By studying the regulation of signaling by hFSHR we hope to learn more about the receptor function and identify new ways to modulate the receptor for novel approaches to contraception or treatment of infertility.
Evolutionary Medicine: Expanding the Insights from Animal Models
Authors: Uhl Elizabeth W.
Published: November 2023

An evolutionary perspective is increasingly being embraced in medicine and is now considered essential for a comprehensive understanding of disease. This perspective has important potential applications in both basic biomedical research and drug development as much of what is known about human disease mechanisms has been learned from the study of animal models. However, since animal models have been developed almost solely based on their similarities to human clinical and tissue pathology, the evolutionary influences determining animal disease phenotypes and why and how they are different from the human have rarely been considered. Because of this focus on similarities, animal models have not been utilized to their full potential. This has contributed to a serious ‘prediction problem,’ which is the failure of animal studies to suitably predict human responses. Another important factor in the prediction problem is that the current disease definitions do not reflect the genetic/molecular complexity underlying disease phenotypes. Diseases have predominantly been classified and studied based upon observable findings including: clinical signs and symptoms, pathology, and response to treatment. Currently advances in technology are allowing genotypic and bioinformatic information to also be incorporated into disease definitions. While this shifting paradigm is leading to a more evolution‐based approach to medicine that will ultimately enhance our understanding of what constitutes ‘health’ and ‘disease’, it is also having a major impact on how animal models are developed and studied. For example, several (in mice often numerous), genetic/molecular changes have been found to produce the same clinical signs and tissue pathology in animals and humans. An important implication of this is that many animal models are more uniform in mechanism than the human diseases they are modeling, and treatments effective in animal models should ideally be tested in the subset of human patients affected through the same genetic/molecular disease mechanisms, not all those with the disease phenotype. Thus, some treatments developed using animal models that fail in the human general disease population and are abandoned may not necessarily be ineffective, while others that are ineffective in the animal models might have efficacy in humans. Evolutionary medicine provides the perspective needed to fully develop the potential and improve the predictive value of animal models. In particular the development of a cross‐species, evolution‐based understanding of disease would improve diagnoses and identify novel therapeutic and preventive interventions for both human and veterinary medicine.
Machinery Mediating Kiss‐and‐Run Mechanism of Cell Secretion
Authors: Jena Bhanu P.
Published: November 2023

Secretion is a fundamental cellular process in living organisms, from yeast to cells in humans. Since the 1950's, it was believed that secretory vesicles completely merged with the cell plasma membrane during secretion. While this may occur, the observation of partially empty vesicles in cells following secretion suggests the presence of transient or so called ‘kiss‐and‐run’ mechanism that allows fractional discharge of intra‐vesicular contents during secretion. This proposed mechanism is mediated by a supramolecular cup‐shaped structure at the plasma membrane called ‘porosome’, which enable secretory vesicles to transiently fuse with the plasma membrane, expel a portion of its contents, and disengage. Porosomes range in size from 15 nm in neurons and astrocytes, to 180 nm in endocrine and exocrine cells. Neuronal porosomes are composed of nearly 40 proteins, compared to the 120 nm nuclear pore complex of >500 protein molecules. Porosome structure, its chemical composition, and functional reconstitution into artificial lipid membrane, and the molecular assembly of membrane‐associated t‐SNARE and v‐SNARE proteins in a ring or rosette complex to establish the fusion pore at the porosome base, and the molecular mechanism of secretory vesicle volume increase required for intravesicular content release during cell secretion, collectively provide a molecular understanding of cell secretion, resulting in a paradigm‐shift in our understanding of the process.

Support or Funding Information

Work presented in this article was supported in part by the National Science Foundation grants EB00303, CBET1066661 (BPJ)
Investigation of a Mitochondrial Twin Arginine Transport Pathway in Arabidopsis thaliana
Authors: Weerakoon Tasmeen Shiny , Ma Qianqian , Dabney‐Smith Carole
Published: November 2023

Most proteins found in organelles such as chloroplasts or mitochondria are synthesized at a location different to their site of function. Cells therefore, must have efficient protein routing processes to allow proteins to go from their site of synthesis to their site of function, which may involve crossing multiple membrane systems. One such vital protein transport pathway is the Tat (Twin Arginine Translocation) pathway. The Tat pathway has been found in bacteria, archaea and eukaryotic organelles such as the chloroplast. The chloroplast Tat pathway (or analogous Tat pathways in bacteria) is capable of transporting fully folded proteins across membranes using only a proton motive force instead of ATP as an energy source. It is composed of three protein components, namely cpTatC, Tha4 and Hcf106 that work together to allow the transport of precursor proteins containing an obligate twin arginine motif in the targeting sequence. However, recent experiments using Nicotiana tabacum cells showed an up‐regulation of a mitochondrial gene coding for a putative mtTatC protein when the cells were subjected to salicylic acid treatment, which mimics a defense response in the plant to attack by pests. We have confirmed a similar upregulation under salicylic acid stress of the putative mitochondrial tatC gene in Arabidopsis thaliana. The presence and upregulation under stress of the mttatC gene suggests the possible presence of a mitochondrial Tat pathway. However, single nuclear genes encode the other cpTat components, which suggests either of the other components would likely dual localize to both organelles when the mtTat pathway was functional. To test for this, we have transformed Arabidopsis thaliana with Tha4‐GFP constructs and looked for its localization in the presence or absence of various biotic or abiotic stresses. Furthermore we are investigating mtTatC and Tha4 protein expression levels at different concentrations of salicylic acid in whole leaf tissue extract and in isolated mitochondria. Evaluating the presence of a mitochondrial Tat pathway will shed light on a unique protein sorting and transport mechanism in plant mitochondria, a facility previously thought to be lost during the mitochondrial evolutionary process.

Support or Funding Information

Dr. Dabney‐Smith Group, Center for Bioinformatics and Functional Genomics (CBFG, Center for Advanced Microscopy and Imaging (CAMI), DOE Office of Science Early Career Research Program (DE‐FG02‐10ER16138)
System xc− Regulation: Effects of Mutations on Membrane Expression and Ubiquitination Status
Authors: Schmidt Mackenzie , Gibson Amanda , Hardy Emma , Versluis Philip , Lang Sara , Georges Anne , Chase Leah
Published: November 2023

System xc− exchanges intracellular glutamate for extracellular cystine across the membrane of many cell types, including astrocytes. Its activity directly regulates the synthesis of the antioxidant glutathione and the extracellular concentration of glutamate in some areas of the brain. Dysregulation of the transporter can lead to excessive glutamate release and excitotoxic cell death or the depletion of glutathione stores and the development of oxidative stress. We recently demonstrated that oxidants acutely upregulate System xc− activity by triggering the rapid redistribution of the transporter from intracellular compartments to the cell surface. Our current work suggests that the trafficking of the transporter may be regulated by ubiquitination and that oxidant exposure directly influences the ubiquitination of the transporter. Since increased ubiquitination tends to decrease the cell surface expression of many membrane transporters, we sought to test the hypothesis that System xc− is ubiquitinated and that the ubiquitination status of the transporter regulates both its cell surface expression and activity. We have used a mutagenesis approach to disrupt putative ubiquitination sites and a putative ubiquitin ligase binding site within a myc‐tagged System xc− construct so that we can understand the role ubiquitination plays in regulating the cell surface expression of System xc−. There are seven highly conserved lysine residues within xCT that are located on the cytoplasmic side of the membrane. These residues are located at positions 4, 37, 41, 43, 422, 472, and 473. We have created mutant forms of this construct containing single or multiple lysine to arginine mutations so that we could assess the effect of these mutations on cell surface expression of System xc‐. Using biotinylation assays and immunocytochemical analysis, we have demonstrated that mutation of the N‐terminal lysine residues increases the cell surface expression of the transporter. We are currently assessing the ubiquitination status of these mutant transporters to determine if the changes in ubiquitination of the transporter are associated with changes in the cell surface expression and activity of the transporter. In addition, we have identified a putative ubiquitin ligase binding site in the C‐terminus of the transporter. Disruption of this binding site also leads to an increase in cell surface expression of the transporter. Collectively, these data suggest that System xc− is regulated by changes in its ubiquitination status such that factors which lead to diminished ubiquitination will allow for increased cell surface expression of the transporter.

Support or Funding Information

This work was supported by RUI 0843564.
Optimization for Transfection and Isolation of C‐Mannosyltransferase DPY19‐L1
Authors: Murdock Brandon
Published: November 2023

Glycosyltransferases are a unique class of enzymes that catalyze the attachment of sugar molecules to proteins as posttranslational modifications. The three ways in which sugar moieties are attached to proteins are by N‐linked, O‐linked, or C‐linked glycosylation. The mannosyltransferase DPY19‐L1 is a mammalian enzyme that attaches a mannose sugar to the second carbon of the indole ring of tryptophan residues in a tryptophan‐x‐x‐tryptophan consensus sequence.

The DPY19‐L1 mannosyltransferase has never been isolated, which is one of our main goals. To accomplish this task, a HEK‐293 cell line was transfected with purified DPY19‐L1 plasmid utilizing cationic lipids to facilitate the uptake of the purified plasmid. Synthesized linear polyethylenimine (PEI) was the cationic lipid used due to its cost efficiency and ease of synthesis. A number of transfection conditions were screened to determine that efficiency was optimized at high confluence and a PEI to DNA ratio of 7:1. Once the protein had been produced, the enzyme was isolated utilizing a his‐tag (hexahistidine tag) on the N‐terminus of the C‐mannosyltransferase. The his‐tag facilitated binding of the protein to the nickel lining of the column during affinity chromatography. The purity of the isolated protein was then verified using SDS‐PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

The long‐term purpose of this research is to determine the enzymatic mechanism by which DPY19‐L1 catalyzes the covalent linkage of mannose to tryptophan residues. For this, reverse phase HPLC (high performance liquid chromatography) will be utilized to determine the enzymatic activity of DPY19‐L1 by measuring the rate of product formation. Subsequently, the residues in the enzyme active site will be analyzed to determine the mechanism by which tryptophan residues are mannosylated.
Cellulase‐Polymer Bioconjugates to Improve Enzyme Stability for Biofuel Production
Authors: Stewart Jamie , Wright Thai , Schmitz Benjamin , Williams Cameron , Makaroff Katherine , Konkolewicz Dominik , Page Richard C
Published: November 2023

Protein polymer hybrids are bioconjugates that offer myriad customizations. The synthesis of these molecules allows for the opportunity to enhance proteolytic stability, manipulate the pH and temperature optimums of enzymes, and construct new and exciting biomaterials. To meet the increasing demands for biofuels, including mandates set by the US Renewable Fuel Standard II (RFS2), new enzymes with improved functionality and stability are desperately needed. Chief among these needs is the RFS2 directive that sets a target of increasing bioethanol production 600‐fold by 2022 and sets a cap on bioethanol that can be produced from grains. In contrast, cellulosic bioethanol produces glucose through enzyme‐mediated degradation of cellulose to produce a precursor that can be used for ethanol production by fermentation. Cellulosic bioethanol production therefore stands as an opportunity to capitalize on an under‐utilized resource, cellulose, to produce bioethanol. To increase the rate of cellulosic bioethanol production we have utilized the cellulase FnCel5a from thermophilic Fervidobacterium nodosum. We have produced cellulase‐polymer conjugates with improved chemical stability. Utilizing site‐directed mutagenesis we control the location of the polymer placement. Herein we present biochemical and biophysical analyses of cellulase‐polymer conjugate stability and activity conferred by interactions between the cellulase and conjugated dimethylacrylamide‐ or acrylamide‐based polymers. Our research seeks to achieve cost‐effective mass production of glucose from cellulose. As the demand for renewable energy increases, the need for rapid and efficient production of this renewable fuel source precursor is critical.

Support or Funding Information

The authors acknowledge financial support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP).
Electrostatic Contributions of Aromatic Residues in Protein‐Carbohydrate Interactions
Authors: Early Stephen A , Diehl Roger C , Kiessling Laura L
Published: November 2023

Protein‐carbohydrate interactions play critical recognition and signaling roles in a diverse array of biological processes—from stem cell differentiation to immune responses. However, the intermolecular forces that govern protein‐carbohydrate interactions have not been quantitatively elucidated. Given the dogma in the field and our data, the goal of this study is to understand the molecular basis for aromatic‐carbohydrate interactions. Previous bioinformatic analyses have shown that aromatic residues are markedly conserved and overrepresented in carbohydrate binding sites while aliphatic residues are dramatically underrepresented. Unlike aliphatic residues, aromatic amino acids present electronegative π‐electron systems that can interact with carbohydrate C–H bonds through CH–π interactions. We posited that if electrostatic contributions are important for CH–π interactions in protein–carbohydrate complexes, differences in the electronics of the aromatic systems and carbohydrate C–H bonds would determine participation in such interactions. We therefore sought to design a system to interrogate the molecular basis for aromatic‐carbohydrate interactions within proteins. Using the immunologically relevant lectin, galectin‐3, the stability and lactose binding affinity of galectin‐3 variants at CH‐π acceptor residue, W181, were assessed through differential scanning fluorimetry and isothermal titration calorimetry. We observed that the replacement of W181 with the smaller aromatic residues phenylalanine, tyrosine, and histidine diminished both protein stability and binding affinity, while replacement of W181 with an aliphatic methionine residue maintained protein stability but abrogated lactose binding. Supported by stability and binding data for the galectin‐3 variants, we conclude that CH‐π interactions are particularly important in promoting the stability of carbohydrate binding proteins as well as their affinity for binding β‐galactosides through the contribution of electrostatics versus hydrophobic interactions.

Support or Funding Information

This work was supported by a UW‐Madison L&S Honors Welton Undergraduate Research Fellowship.
Identification of a post‐translational modification with ribitol‐phosphate and its defect in muscular dystrophy: Roles of ISPD, fukutin, and FKRP in α‐dystroglycan glycosylation
Authors: Kanagawa Motoi , Kobayashi Kazuhiro , Tajiri Michiko , Manya Hiroshi , Kuga Atsushi , Yamaguchi Yoshiki , Wada Yoshinao , Endo Tamao , Toda Tatsushi
Published: November 2023

Glycosylation is an essential post‐translational modification that underlies many biological processes and diseases. α‐dystroglycan (α‐DG) is a receptor for matrix and synaptic proteins that causes muscular dystrophy and lissencephaly upon its abnormal glycosylation (α‐dystroglycanopathies). Here we identify the glycan unit ribitol 5‐phosphate (Rbo5P), a phosphoric ester of pentose alcohol, in α‐DG. Rbo5P forms a tandem repeat and functions as a scaffold for the formation of the ligand‐binding moiety. We show that enzyme activities of three major α‐dystroglycanopathy‐causing proteins are involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain‐containing (ISPD) is cytidine diphosphate ribitol (CDP‐Rbo) synthase. Fukutin and fukutin‐related protein are sequentially acting Rbo5P transferases that use CDP‐Rbo. Consequently, Rbo5P glycosylation is defective in α‐dystroglycanopathy models. Supplementation of CDP‐Rbo to ISPD‐deficient cells restored α‐DG glycosylation. These findings establish the molecular basis of mammalian Rbo5P glycosylation and provide insight into pathogenesis and therapeutic strategies in α‐DG‐associated diseases.

Support or Funding Information

Japan Society for the Promotion of Science 15H04352
A Novel Hemolysin with Anti‐cancer and Anti‐fungal Properties Binds to Serum Glycoproteins and Cholesterol
Authors: Welch Christina , Fan Ni , Brown Robert , Talaga Melanie , Fueri Ashli , Driscoll Kyle , Lawry Kevin , Vizurraga Alexander , Rekhi Ramandeep , Bandyopadhyay Purnima , Dam Tarun
Published: November 2023

Lysins are capable of disrupting mammalian cell membranes by specifically binding to membrane components. Lysin can be a protein, a peptide or a glycoside. Certain lysins, called hemolysins, can disintegrate mammalian erythrocytes. A few of the reported lysins can bind to specific glycan structures. Here we report, for the first time, a glycan‐binding hemolysin (named Hemolysin‐X or HelyX) from a flowering plant. HelyX was purified by ammonium sulfate precipitation, size exclusion chromatography and high performance liquid chromatography (HPLC). Activity and ligand binding properties of HelyX were studied by plate based assays and spectrophotometric analysis. Interactions of HelyX with mammalian and fungal cells were studied through microscopy and flow cytometry. HelyX showed rapid and robust hemolytic activities even at a very low concentration. The molecular weight of this hemolysin is 2 kDa. Hemolysis by HelyX was completely inhibited by serum glycoproteins (Fetuin, asialofetuin, thyroglobulin and fibrinogen) and cholesterol. Interestingly, the hemolysins of cholera bacteria were reported to show similar binding specificities. When tested with colon and breast cancer cell lines, HelyX showed significant apoptotic activities. The hemolysin also inhibited the growth of yeast cells.

Support or Funding Information

Michigan Technological University