bluuebunny/crossref_metadata_2025 huggingface dataset:
A Regulatory Mechanism for Nuclear Lipid Droplet Biogenesis
Authors: Lee Jonghwa , Salsman Jayme , Dellaire Graham , Ridgway Neale
Published: June 2021

Lipid droplets (LDs) are metabolic energy reservoirs that store and release fatty acids in response to nutrient and hormonal signals. Cytosolic LDs (cLDs) are thought to form in the endoplasmic reticulum (ER) by a process that requires a coordinated synthesis of triglycerides (TG) and phosphatidylcholine (PC), the primary phospholipid component of the surface monolayer that stabilizes the neutral lipid core. Nuclear LDs (nLDs) are present in many eukaryotic cells, suggesting a related mechanism for nLD assembly that involves de novo phospholipid and TG synthesis on the inner nuclear membrane (INM). The rate‐limiting enzyme for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα), is activated by translocation to the INM and nLDs. As well, nLDs and CCTα associate with promyelocytic leukemia nuclear bodies (PML‐NBs). PML‐NBs are dynamic sub‐nuclear structures involved in regulating several important biological processes, including gene expression and oxidative stress response. The storage of TG in nLD/PML‐NB/CCTα complexes could buffer the nucleus against fatty acid overload and detect and respond to lipid stress via PML‐NB‐associated signaling pathways. In this study, we investigated how the nLD/PML‐NB/CCTα complex is formed during oleate treatment of human U2OS osteosarcoma cells and Caco2 colorectal adenocarcinoma cells. Super‐Resolution Radial Fluctuation (SRRF) imaging revealed that PML proteins and CCTα co‐localized in a bead‐like ring around the nLD. Quantitative fluorescence microscopy revealed that PML‐NBs and CCTα were localized on larger nLDs in both U2OS and Caco2 cells. Knocking out PML expression by CRISPR/Cas9 in U2OS cells (PML‐KO) resulted in fewer and smaller nLDs, and the remaining nLDs had a 40% reduction in associated CCTα. The size of CCTα‐positive nLDs in control and PML‐KO cells was comparable. In contrast, CRISPR/Cas9 knockout of CCTα in Caco2 cells (CCTα‐KO) increased the size and decreased the number of LDs, resulting in increased association of PML‐NBs with large nLDs. Collectively, these data suggest that PML‐NBs and CCTα are required for regulating the size and number of nLDs in response to exogenous oleate. Fatty acids promote the formation of large nLDs that interact with PML‐NBs. This interaction recruits CCTα to potentially stimulate CCTα activity and PC biosynthesis required for the surface monolayer.

Support or Funding Information

Canadian Institutes of Health Research

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of a novel small‐molecule activator that corrects G6PD deficiency
Authors: HWANG SUNHEE
Published: June 2021

Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic disorders, is caused by over 160 different point mutations and results in a variety of health problems, including hemolytic anemia and neurological damage. As G6PD is a major source of protective anti‐oxidant through NADPH production, G6PD deficiency likely contributes to the severity of many other acute and chronic diseases associated with increased oxidative stress. However, no medications are available to treat G6PD deficiency directly; thus, we sought to identify a small molecule that increases catalysis of G6PD mutant enzymes. Crystal structure and mutagenesis analyses of one common mutant (Canton, R459L) provided insight into the functional defect of the enzyme. Using high‐throughput screening, we identified AG1, a small molecule that increases the activity and/or stability of the wild‐type, the Canton mutant and several other common human G6PD mutants as well. AG1 reduced oxidative stress and increased NADPH levels in vivo, in zebrafish, and prevented chloroquine‐induced hemolysis in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the clinical problems associated with multiple variants of G6PD deficiency.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Generation and characterization of a myeloid cell‐specific Cyp19a1 knockout mouse model
Authors: Han Weiguo , kovalchuk Nataliia , Ding Liang , Liu Pengfei , Yang Weizhu , Zhang Qing‐Yu , Ding Xinxin , Winkle Laura
Published: June 2021

CYP19A1 (the aromatase) is a key enzyme in the biosynthesis of estrogens. Estrogens have important regulatory functions, including roles as a major mitogenic stimulus for cellular proliferation. It has been proposed that macrophage CYP19A1 can increase estrogen production at sites of tissue injury, thereby influencing the balance between normal tissue repair and pathogenic outcomes such as hyperproliferation and oncogenic transformation following xenobiotic‐induced toxicity. Our goal is to test this hypothesis using a cell‐specific Cyp19a1‐null mouse model. Here we report the generation and initial characterization of a myeloid cell (including macrophage) specific Cyp19a1‐null mouse model. A newly generated Cyp19a1‐lox mouse was intercrossed with a Cre‐transgenic mouse for myeloid cell specific deletion of a critical CYP19A1 exon. Cyp19a1 deletion was confirmed by PCR analysis of genomic DNA isolated from macrophages in lung bronchoalveolar lavage fluid; according to results of a quantitative PCR analysis, the deletion efficiency was greater than 90% in these cells. Expression of CYP19A1 protein was detected in alveolar macrophages from wild‐type mice, but not in macrophages from Cyp19a1‐null mice, by immunohistochemical and immunofluorescence staining with an anti‐CYP19A1 antibody. Identity of macrophages was confirmed by staining with an anti‐CD14 (macrophage biomarker) antibody. The ability of macrophages in lung bronchoalveolar lavage fluid from wild‐type mice to synthesize estradiol from testosterone was demonstrated using in vitro assays and LC‐MS based detection of estradiol. The aromatase activity was not detected in macrophages from the null mice. Tissue specificity of Cyp19a1 deletion was demonstrated by the lack of detection of the null allele by a quantitative PCR analysis of genomic DNAs from multiple other sources, including lung, liver, ovary, testis, and tail. Homozygous null mice are fertile and show normal growth and activity. They are currently studied for sensitivity to the lung toxicity of naphthalene, a ubiquitous pollutant and possible human lung carcinogen.

Support or Funding Information

Supported in part by NIH grant ES020867

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Control of the Ca2+‐Mediated Endothelial Cell Migration by Connexin 43‐Formed Hemichannels
Authors: AMADOR HILDA ESPINOZA , CORDOVA ALEXANDRA , FIGUEROA XAVIER
Published: June 2021

Endothelial cell migration is a key process in endothelium repair, tube formation and angiogenesis. The initiation and progress of endothelial cell migration is orchestrated by the generation of coordinated Ca2+ signals through Ca2+ release from the intracellular stores and Ca2+ entry. However, the molecular pathways involved in the Ca2+ influx have not been determined. Connexins (Cx) play a central role in the coordination of endothelial cell function, directly by cell‐to‐cell communication via gap junction and, indirectly, by the release of autocrine/paracrine signals through Cx‐formed hemmichannels. In addition to the release of signaling molecules, connexin hemichannels are permeable to Ca2+ and, thus, the opening of these channels may contribute to the Ca2+ influx observed during endothelial cell migration. Endothelial cells mainly express Cxs 37, 40 and 43 and, of these three connexin proteins, Cx43 has been reported to form hemichannels in endothelial cells. Interestingly, caveolae seems to be involved in the coordination of the Ca2+‐mediated endothelial cell migration and Cx43 has been found to be associated with caveolin‐1, a structural protein of the caveolae. However, the participation of Cx43 in endothelial cell migration has not been determined. Primary cultures of mesenteric endothelial cells were used to evaluate cell migration by the wound healing assay. Cx43 and caveolin‐1 distribution was assessed by immunofluorescence, activation of Cx hemichannels by measuring ethidium uptake and changes in intracellular Ca2+ concentration [Ca2+]i by loading the cells with Fluo‐4. The scratch of the endothelial cell monolayer with a micropipette tip induced a strong increase in [Ca2+]i and ethidium uptake by cells of the migration front, but not those of the monolayer. Treatment with 18‐b‐Glycyrrhetenic acid (bGA, 50 μM), a general connexin‐formed channel blocker, 37,43GAP27 (200 μM), a Cx37 and Cx43 blocking peptide, and TAT‐Gap19 (300 μM), a Cx43 hemichannels specific blocking peptide, blunted the increase in [Ca2+]i and, consistent with the participation of Cx43 in this process, 37,43GAP27 also reduced the increment in ethidium uptake. In addition, endothelial cell migration was associated with the re‐localization of Cx43 and caveolin‐1 to the rear part of the cells in the migration front, which was prevented by the treatment with TAT‐Gap19. As expected, bGA and TAT‐Gap19 also inhibited the endothelial cell migration rate. These results indicate that Cx43 hemichannels play a central role in the coordination of the Ca2+ signaling that mediates the endothelial cell migration.

Support or Funding Information

Grants: FONDECYT 1150530 and CONICYT 21170977

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AIFm2 mediated retrograde stress signaling during ischemia‐reperfusion injury in heart: Role of 4‐HNE adduction
Authors: Sharma Sudha , Bhattarai Susmita , Subedi Utsab , Bess Ezra , Bhuiyan Md.Shenuarin , Panchatcharam Manikandan , Miriyala Sumitra
Published: June 2021

Myocardial ischemia‐reperfusion injury results from restoration of normal coronary blood circulation after a prolonged period of circulatory arrest. Oxidative stress is one of the underlying mechanism for the pathogenesis of ischemia‐reperfusion injury. Oxidative stress results from a disturbance in the homeostatic balance between free radicle generation and availability of antioxidant molecule to scavenge them. Manganese superoxide dismutase (MnSOD), mitochondrial based antioxidant is capable of converting superoxide radical (O2•−) into oxygen. Reactive oxygen species cause lipid peroxidation and subsequent production of 4‐hydroxy‐2‐nonenal (4‐HNE) from polyunsaturated fatty acid (PUFA). 4‐HNE can modify signaling by causing adduction of cellular components such as proteins and nucleic acids. Mice lacking MnSOD specifically in cardiomyocyte (Myh6‐SOD2Δ) showed early mortality (~ 4 months) due to mitochondrial dysfunction and subsequent cardiomyopathy. Myh6‐SOD2Δ mice also had decreased mitochondrial respiration and cardiac performance shown by Seahorse XF analyzer and M‐mode echocardiography respectively. Following ischemia‐reperfusion injury, MnSOD expression was reduced shown by Real‐Time PCR array, Immunoblot, and Immunohistochemistry. Immunoblot analysis of AIFm2 showed increased expression in the ischemic heart. Normally AIFm2 localized to mitochondria, however, following ischemia 4‐HNE adducted AIFm2 translocated from mitochondria to nucleus thereby binding to DNA and resultant myocardial apoptosis. With the help of mass spectrometric analysis, it has been shown that His 174 and Cys 187 are two sites of AIFm2 where 4‐HNE adduction occurs. These results identify the role of 4‐HNE adduction in the regulation of mitochondrial retrograde signaling mediator protein apoptosis‐inducing factor associated mitochondrion protein 2 (AIFm2) during myocardial ischemia‐reperfusion injury.

Support or Funding Information

NIH‐ AA025744; UL1RR033173; HL141998;NISBRE‐ COBRE1P20GM121307‐01A1; AAA‐FGAP‐5148; AHA ‐ 0950118G; 10SDG4190036

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial Uncoupling Protein‐2 and Fibroblast Senescence in Age‐Related Lung Fibrosis
Authors: Rangarajan Sunad , Locy Morgan L , Zmijewski Jaroslaw W , Tuder Rubin M , Eickelberg Oliver , Thannickal Victor J
Published: June 2021

Objective

Our objective was to examine the role of mitochondrial uncoupling protein‐2 (UCP2), highly expressed in lung fibroblasts of patients with idiopathic pulmonary fibrosis (IPF), in mediating mitochondrial dysfunction, fibroblast senescence and myofibroblast differentiation in age‐related pathologic fibrosis.

Methods

We studied tissues and fibroblasts obtained from lung explants of IPF patients and utilized the murine model of lung fibrosis and resolution following bleomycin‐induced injury. We silenced UCP2 expression using siRNA in vitro and in vivo. Specific endpoints were analyzed by RT‐PCR, SDS‐PAGE western blotting, immunofluorescence, flow cytometry, mass spectrometry and extracellular flux analysis.

Results

UCP2 gene expression was increased in lungs (whole lung lysates as well as fibroblasts) of IPF patients compared to those without IPF; in particular, mesenchymal stromal cells derived from bronchoalveolar lavage of patients with rapidly progressive clinical course had higher UCP2 expression compared to those with a slower course. Human lung fibroblasts undergoing replicative senescence expressed high levels of UCP2. Lung fibroblasts from aged mice expressed higher UCP2 than younger counterparts, more pronounced after bleomycin‐induced lung injury. UCP2 silencing in IPF fibroblasts decreased pro‐fibrotic markers such as α‐SMA and collagen; this was associated with enhanced mitochondrial biogenesis, decreased mitochondrial ROS (superoxide and hydrogen peroxide), increased ATP‐linked oxygen consumption and decreased aerobic glycolysis. In addition, UCP2 silencing induced a decrease in senescence markers and decreased expression of senescence‐associated secretory phenotype (SASP) genes. In aged mice after bleomycin injury, therapeutic oro‐tracheal administration of UCP2 siRNA resulted in decreased lung collagen and fibrotic remodeling, indicating enhanced resolution capacity.

Conclusion

Our results suggest that expression of mitochondrial UCP2 in lung fibroblasts increases with age. UCP2 is highly expressed in lung fibroblasts of patients with IPF, more so in those with rapidly progressive clinical course; UCP2 promotes myofibroblast senescence and impairs resolution of bleomycin injury‐induced fibrosis in aged mice. Further studies are warranted to study the link between cellular aging and UCP2, and the therapeutic potential of targeting UCP2 in age‐related fibrotic disorders such as IPF.

Support or Funding Information

NIH grants K08 HL135399 (SR); P01 HL114470 and R01 AG046210 (VJT), VA Merit Award I01BX003056 (VJT)

Schematic showing the role of UCP2 in fibroblast (patho)biology

image

Schematic showing the role of UCP2 in fibroblast (patho)biology

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Deubiquitylase USP4 Regulates AQP2 Membrane Targeting and Abundance in mpkCCD Cells
Authors: Murali Sathish K , Moeller Hanne H , Fenton Robert A
Published: June 2021

Aquaporin 2 (AQP2) is a vasopressin regulated water channel in the renal collecting duct responsible for body water homeostasis. AQP2 is poly‐ubiquitylated at a single lysine residue, K270. Ubiquitylation of AQP2 increases its endocytosis and lysosomal degradation, and plays a role in ERAD mediated AQP2 degradation via its interaction with the c‐terminal of Hsp70 interacting protein (CHIP). Our recent proteomics screen of mpkCCD14 (mouse collecting duct) cells found several collecting duct‐specific deubiquitylases (DUBs) that may be important for removing the polyubiquitin chain on AQP2 and thereby modifying its cellular fate. Here, we sought to elucidate if one of these DUBs, the Ubiquitin Specific Peptidase 4 (USP4), has a role in regulating AQP2 function. USP4 was abundant in mouse kidneys and mpkCCD14 cells and could be co‐immunoprecipitated with AQP2 from mouse kidney, suggesting they interact. Several shRNA mediated USP4 knockdown mpkCCD14 cell lines were developed, each with significantly reduced USP4 mRNA and protein levels. USP4 knockdown cells had significantly reduced total and phosphorylated AQP2 protein levels, but not AQP2 mRNA levels, compared to control shRNA cell lines. Vasopressin receptor type 2 levels and dDAVP‐induced cAMP production were similar between control and USP4 knockdown cells, as were expression levels of several other transport proteins including AQP4 and alpha‐ENaC. Cycloheximide chase studies showed a significantly decreased protein half‐life for AQP2 in USP4 knockdown cells, suggesting that USP4 has a role in AQP2 protein stability/degradation. dDAVP‐induced AQP2 membrane accumulation in USP4 knockdown cells was significantly reduced compared to the control cells. Total AQP2 ubiquitylation levels were higher in USP4 knockdown cells compared to control cells, suggesting that loss of USP4 limits AQP2 deubiquitylation. Taken together, our results indicate that USP4 has a major role in modulating AQP2 ubiquitylation and in maintaining normal AQP2 levels and trafficking.

Support or Funding Information

Funding is provided by the Novo Nordisk Foundation, the Lundbeck Foundation and the Danish Medical Research Council.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased activity of the angiotensin converting enzyme C‐domain reduces melanoma growth by stimulating M1 macrophage polarization
Authors: Khan Zakir , Cao Duo Yao , Giani Jorge F. , Bernstein Ellen A. , Veiras Luciana , Fuchs Sebastien , Liu George Y. , Bernstein Kenneth E.
Published: June 2021

ACE is composed of two independent catalytic domains. To investigate the specific role of each ACE domain in tumor resistance, we made transgenic mice which over express in myeloid cells either wild type ACE (Tg‐ACE mice) or ACE lacking N‐ or C‐domain catalytic activity (Tg‐NKO and Tg‐CKO mice). Tg‐ACE and Tg‐NKO mice strongly suppressed the growth of B16‐F10 melanoma due to increased ACE expression by macrophages. In contrast, Tg‐CKO mice resist melanoma no better than wild type animals. The effect of ACE over expression reverts to wild type with an ACE inhibitor but not with an AT1 receptor antagonist. Over expression of the ACE C‐domain in macrophages drives cells towards a pronounced M1 phenotype upon tumor stimulation, with increased activation of NF‐kB and STAT1, and decreased activation of STAT3 and STAT6. TNFα is important in M1 activation of macrophages; TNFα blockade reverted Tg‐NKO macrophages to a wild type phenotype. Increased ACE C‐domain expression induced increased levels of the transcription factor C / EBPβ in macrophages, an important stimulus for TNFα expression. In contrast, there was decreased expression of IL‐4Rα, a key receptor for M2 activation of macrophages. Natural ACE C‐domain specific substrates are not well described. Nonetheless, the peptide(s) responsible for the ACE‐mediated enhancement of myeloid function are substrates and products of only the ACE C‐domain.

Support or Funding Information

This study was supported by NIH grant P01HL129941 (KEB), AI143599 (KEB), R21AI114965 (KEB), R01HL142672 (JFG), P30DK063491 (JFG), AHA grant 17GRNT33661206 (KEB), and AHA grant 16SDG30130015 (JFG).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Localization of Lipid Exchange Machinery to TBC‐ER Contact Sites
Authors: Adams Arlo , Yoo Kang Mu , Vogl Wayne
Published: June 2021

The production of sperm cells in the mammalian testis is complex, and requires a well‐organized restructuring of the epithelium to which late spermatids are attached before their subsequent release at the completion of spermatogenesis. Disassembly of large adhesion like junction complexes called ‘ectoplasmic specializations’ (ESs) is necessary for late spermatids to detach from Sertoli cells. Endocytic structures proposed to disassemble ESs are called ‘tubulobulbar complexes’ (TBCs). TBCs share some similarities with clathrin‐mediated endocytosis (CME) in that they have a clathrin coated pit and a dendritic actin network that likely drives invagination. Unlike CME however, the actin network is not as temporary, and forms a long extended neck that progresses into an actin free portion that forms a swollen bulb, while the structure is still continuous with the Sertoli cell plasma membrane. Using antibody localization techniques, we have previously established the identity of the TBC bulb as an early endosome. Interestingly, the bulbs of TBCs form extensive ER contacts, the function of which is unclear. Recent expansions in our understanding of ER contacts have revealed that they serve at least three important functions with respect to ER‐endosome contacts: (1) positioning and scission of endosomes (2) calcium exchange and (3) lipid transfer. We have previously localized calcium exchange machinery to the TBC‐ER contact site, but never before have lipid transfer machinery been localized here, or to any structures related to the apical processes of Sertoli cells. If lipid transfer occurs at TBC‐ER contacts, then protein machinery known to be involved in these processes should be localized to these sites. Here we use immunofluorescence and STED super resolution imaging to localize known lipid exchange machinery to the ER of Sertoli cell apical processes. Oxysterol binding protein‐related protein 9 (ORP9) was localized to the TBC‐ER contacts and a known ORP interactor and anchor, VAMP‐associated protein A (VAPA), was localized ubiquitously throughout the ER of the apical process, including the TBC‐ER contact. This data is consistent with the hypothesis that lipid exchange is one of the key functions of TBC‐ER contacts.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant awarded to AWV; Alexander Graham Bell Scholarship (NSCERC) awarded to AA

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Hexavalent Chromium Exposure on Global Translation and No‐Go Decay
Authors: Seaberg Lucas , Segal Scott P
Published: June 2021

Chromium IV (Cr VI) is a byproduct of a number of industrial processes, including the manufacture of stainless steel and the protection of power plant cooling towers. Cr(VI) is a potent carcinogen through its ability to oxidize guanine bases, and when they are repaired resulting in G to A transitions in DNA. The ability of Cr(VI) to damage DNA has been well studied. However, the impact of Cr(VI) exposure on the transcriptome, since RNA also contains guanine bases, is not fully understood. Previous studies show that Cr(VI) has the ability to directly damage mRNA through formation 8‐oxo(G) bases, which leads to ribosome stalls during translation. This leads to activation of the No‐Go Decay pathway (NGD), in which Dom34p and Hbs1p act to remove stalled ribosomes, and cleave the mRNA at the ribosome stall site. The 5′ fragment is degraded 3′ to 5′ by the cytoplasmic exosome and the 3′ fragment localizes to P‐bodies and is degraded by the 5′ to 3′ exonuclease Xrn1p.

Currently, little is known about which mRNA are subject to Cr(VI) mediated oxidative damage, and subsequent NGD. In order to elucidate which mRNA are being damaged by Cr(VI) and subject to NGD, dom34Δ; xrn1Δ and dom34Δ; ski2Δ double mutant strains were created. These strains were designed in order to shut off either 5′ to 3′ or 3′ to 5′ degradation, allowing for analysis of damaged transcripts. Growth of double mutant strains was observed on both nutrient‐rich media and media containing Cr(VI). Growth of the dom34Δ; xrn1Δ strain was inhibited when plated on media containing Cr(VI). The dom34Δ; xrn1Δ strain compared closely to dom34Δ growth in nutrient rich and oxidizing conditions. When compared to xrn1Δ under both growth conditions, the dom34Δ; xrn1Δ strain appeared healthier. Along with this, P‐body assembly during oxidative stress will be observed to better characterize the strain's response to Cr(VI) exposure.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutational analysis of interdomain binding interface of Grb7
Authors: Biswas Ashis Kumar
Published: June 2021

Grb7 is a member of the Grb7 adaptor protein family (Grb7, Grb10 and Grb14) and is a suspected promoter of tumor progression. The multi‐domain Grb7 protein is composed of the Proline‐Rich (PR), Ras‐associating (RA), Pleckstrin Homology (PH), and Src Homology 2 (SH2) domain[Office1] s. Grb7 is observed over‐expressed, along with the receptor tyrosine kinase ErbB2, in 20–30% of breast cancer cells, and is involved in increased cell migration and formation of metastases [1]. It is believed the RA, PH and SH2 domains interact with various downstream signaling partners to potentiate Grb7's role in cell migration [1]. Grb7 undergoes intermolecular homo‐dimerization through its SH2 domain [2] while also undergoing an intramolecular domain interaction via its SH2 and RA‐PH domains [3]. These interactions are believed to affect the Grb7 protein functional state. Based upon our previous results substantiating the Grb7 intramolecular domain interaction, we now seek to characterize the binding interface between the Grb7 RA‐PH, and Grb7 SH2 domains. Our approach is to use mutational analysis to target the RA‐PH and SH2 domain interfaces we hypothesize may be involved in the intramolecular interaction. At present we have successfully produced the SH2 domain mutants V45A, K46R, D47E and S48A by overexpression in a bacterial system and GST fusion‐protein based purification. We have also expressed and purified the Grb7 RA‐PH two‐domain construct by a method previously developed in our laboratory. With these protein domains in hand we will perform biophysical studies to probe the interaction between the Grb7 RA‐PH, and SH2 domains. Proposed experimental approaches for this study include isothermal titration calorimetry, surface plasmon resonance, nuclear magnetic resonance, circular dichroism, and dynamic light scattering. These studies will help us to better understand how Grb7 functions in tumor progression, and further our ultimate goal of designing Grb7‐based therapeutics for treating aggressive breast cancers.

[Office1]You can't use acronyms in an abstract without first defining them. Your reader will get lost.

Support or Funding Information

NIH

5‐SC3‐GM116728

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deciphering the role of N‐terminal methylation in modulating yeast protein function including the multitasking stress response protein, Hsp31
Authors: Chen Panyue , Dong Guangping , Hazbun Tony , Huang Rong
Published: June 2021

The first cytosolic N‐terminal methyltransferase identified in yeast, Tae1, recognizes mainly a protein N‐terminal motif sequence of M‐X‐P‐K. This conserved sequence motif is found in multiple proteins including ribosomal proteins, histones and small heat shock proteins across species. The human homolog of Tae1, N‐terminal methyltransferase 1 (NTMT1/NRMT1) has important functions in cell division by regulating RCC1 and centromere proteins such as CENP‐A and is implicated as a cancer target. Although these studies have been informative, the biological function of alpha N‐terminal methylation in yeast and human remains largely unknown. Our research is directed at unravelling the role of N‐terminal methylation in regulating yeast protein function and is currently focused on Hsp31, a multifunctional chaperone and enzyme involved in oxidative stress response. We are also investigating N‐terminal methylation on a proteome‐wide basis.

Immuno‐detection and tandem mass spectrometry (MS/MS) assay with overexpressed Hsp31‐ MORF or physiologically expressed Hsp31‐GFP purified from WT and Tae1 deficiency strains demonstrated that Hsp31‐MORF is methylated at the first alanine (initial methionine is efficiently removed by a native methionine aminopeptidase) after the protein is synthesized. We also observed that the methylation types are exclusively mono‐ or di‐ methylation, with no tri‐methylated species observed. This is the first demonstration that the Hsp31 chaperone protein is N‐terminally methylated. Interestingly, methylation of Hsp31 was decreased but not completely eliminated in Tae1 deficient strains. This is the first evidence of such an overlapping methyltransferase activity which suggests the presence of another enzyme with N‐terminal methyltransferase activity. It unclear how the biological function of Hsp31 is affected by methylation and the difference between mono‐/di‐ methylation and tri‐methylation. We also verified N‐terminal methylation on other potential Tae1 substrates by MS/MS, including Hsp33 and a xylose and arabinose reductase. We conclude that N‐terminal methylation occurs with diverse substrates and that yeast is an excellent model to probe the function of this post‐translational modification. Further investigation is ongoing to determine if and how N‐terminal methylation modulates Hsp31 functions using site‐directed mutagenesis and biological activity assays.

Support or Funding Information

Funding: Purdue University MCMP Research Enhancement 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.
Design and Synthesis of a Photoisomerizable Second Generation Kinase Inhibitor
Authors: Allison Jessica , Kazmierczak Marcin , Streu Craig
Published: June 2021

BCR/ABL is a chimeric oncogene made up of breakpoint cluster region protein (BCR) from chromosome 22 and Abelson murine leukemia (ABL) from chromosome 9. This genetic recombination results in a shortened chromosome 22, which is also known as the Philadelphia Chromosome. The BCR/ABL fusion protein produced as a result of this translocation mutation causes the resulting tyrosine kinase enzyme to become constitutively active. Given the critical regulatory role of ABL in cell division, its constitutive activation results in uncontrollable cell division, which in this case, most often leads to chronic myeloid leukemia (CML). BCR/ABL has also been associated with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

The first‐generation treatments for diseases caused by this mutation were approved for use in 2001 and were kinase inhibitors. These inhibitors effectively shut off the dysfunctional kinase and shut down uncontrolled cell division. Over time, cancer cells can become resistant to drugs and many CML patients begin to experience resistance to first‐generation therapies. These resistances are caused by mutations in the target kinase, which change the drug binding site and/or protein conformation. Although the development of second‐generation therapeutics has addressed many of these issues, one common problem remains off‐target interactions of these drugs, which result in undesirable side effects. One way to address both of those issues is create drugs that can be switched on/off when and where necessary. Light can be used to change the shape of molecules. Since the shape of molecules is important for their binding to a biomolecule, changing shape can change bioactivity. The purpose of this research is to test the photo‐induced bioactivity of azologues of a second‐generation BCR/ABL inhibitor. Azologues are light activated versions of known biologically active molecules, whose photoisomerizable properties are imparted by the presence of an azo‐stilbene group. Compounds that can be selectively spatially and temporally activated by light could circumvent these off‐target interactions. Additionally, photoisomerizable molecules can be useful tools for investigating biological systems. Given the spatial and temporal control that photoisomerization allow, they may be utilized as powerful chemical tools for the study of time and location dependent cellular processes. The design and synthesis of the target molecule will be outlined as well as the unique synthetic challenges associated with this class of biomolecule.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An energetics database to model protein‐protein interactions
Authors: McFadden Collin Michael , Anderson Samantha , Senes Alessandro
Published: June 2021

The human genome contains approximately 2,300 single‐pass membrane proteins, and any two proteins can form a dimer with millions of different conformations. Our understanding of the stability and energetics of these membrane proteins informs the study of various signaling complexes, receptor tyrosine kinases, and immunological complexes. This project creates a tool that uses pre‐computed van der Waals and hydrogen bond interaction data for various amino acid combinations in an array of orientations to model the optimal conformation for any pair of sequences. Our lab showed when two proteins are identical, forming a homodimer, their interaction is stabilized primarily by hydrogen bonds and van der Waals forces. Our current algorithm can accurately predict the structure of symmetric homodimers, but in cases when the two protein sequences differ, additional geometric variation must be considered. This project expands our capabilities to include interactions with two different sequences and asymmetric orientations. This will help elucidate the functionality of uncharacterized membrane proteins and predict the likelihood that a helix interacts with other proteins in the membrane. The algorithm will also estimate the stability of different orientations of the same pair of helices and quantify the likelihood that the two proteins would dimerize. Due to the physical limitations of observing membrane proteins, computational methods and computer simulations improve the resources available for studying signaling interactions in membrane proteins and supports in vitro experiments on these 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.
Induction of a Non‐Apoptotic Cell Death in Momordica Charantia ‐Treated Human Cancer Cell Lines
Authors: Ehigie Adeola Folasade , Wei Peng , Wei Taotao , Ehigie Leonard Ona , Yan Xiyun , Ojeniyi Fiyinfoluwa Demilade , Olorunsogo Olufunso Olabode
Published: June 2021

One major mechanism of death in cancer cells is via the induction of apoptosis, the best‐studied form of programmed cell death. Although apoptosis is essential in many normal physiological processes, including tissue sculpting during embryogenesis, development of the immune system and destruction of damaged cells, its alteration is a major cause of cancer. Having confirmed the cytotoxic effect of certain fractions derived from the crude water‐soluble extract (CWSE) of Momordica charantia Linn (MC) in a previous study (Ehigie et al., 2016), we investigated in this study, the mode of death induced by these fractions, both in human breast [MDA‐MB‐436] and lung cancer [A549] cell lines, using PI (Propidium Iodide), DAPI (4,6‐diamidino‐2‐phenylindole dihydrochloride) and Caspase‐3 Activation Determination Assays. Western blotting was also employed to verify the activation of caspase‐3 and the mitochondrial release of cytochrome c. The ethyl acetate (D3), n‐hexane (D4) and dichloromethane (D5) fractions were all obtained from the CWSE via solvent partitioning and then administered at 100 and 125 μg/mL for 24 h. Though we observed that the cancer cells treated with the ethyl acetate fraction of the CWSE of M. charantia were the most killed, we saw neither the evidence of chromatin condensation, cell shrinkage and apoptotic bodies, nor the activation of caspase‐3 or cytochrome c release! Data obtained for caspase‐3 activation by MC show no significant (P<0.05) difference between the negative control and all MC‐treated groups for both cell types with the negative control group for MDA‐MB 436 cells at 90.97±9.03 compared with D3‐treated (89.26±3.17 and 96.29±3.85), D4‐treated (90.99±19.78 and 91.71±4.83) and D5‐treated (93.95±2.95 and 88.91±1.00) cells at 100 and 125 μg/mL respectively, similar to values obtained for A549 cells; negative control (69.69±2.60) compared with D3‐treated (70.39±6.93 and 76.21±2.65), D4‐treated (73.67±1.32 and 74.25±0.00) and D5‐treated (71.80±3.26 and 71.17±14.85) cells at 100 and 125 μg/mL respectively. From the foregoing, we conclude that the cell death induced by the ethyl acetate, n‐hexane and dichloromethane fractions of MC in the selected cancer cell lines is by a non‐classical apoptotic mechanism.

Support or Funding Information

Organization for Women in Science for the Developing World (OWSD) and Swedish International Development Cooperation Agency (SIDA)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insights into the Role of the Membrane on PLCβ and Gαq‐Mediated Activation and Adsorption
Authors: Hudson Brianna N. , Lyon Angeline M.
Published: June 2021

Phospholipase C β (PLCβ) enzymes are peripheral membrane proteins that are required for normal cardiovascular function and whose dysregulation results in cardiovascular disease. PLCβ hydrolyzes phosphatidylinositol‐4,5‐bisphosphate (PIP2) to produce the two potent second messengers inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG), increasing intracellular Ca2+ and activating protein kinase C. PLCβ has low basal activity, but is activated downstream of G protein‐coupled receptors (GPCRs) through direct interactions with the heterotrimeric G protein subunits Gαq and Gβγ. While G proteins stimulate activity, they are insufficient for full activation, suggesting the membrane contributes to full activation. However, the molecular basis for the role of the membrane in basal and G‐protein stimulated activity are not well understood. We seek to understand how membrane composition helps regulate adsorption of PLCβ to the membrane, and how interfacial activation contributes to basal activity and Gαq‐dependent activation. Using a model membrane system, we are applying an innovative combination of atomic force microscopy, mass spectroscopy, and biochemical assays to begin understanding how the membrane itself, regulatory elements within PLCβ, and Gαq regulate lipase activity. These studies provide the first structure‐based approach to understanding how the cell membrane regulates the activity of this essential effector enzyme.

Support or Funding Information

This work was supported by the Purdue Center for Cancer Research, the Showalter Foundation, American Heart Association Scientist Development Grant 16SDG29920017, and NIH 1R01HL141076‐01 to A. Lyon and a Purdue Doctoral Fellowship to B. Hudson.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of the proteoglycan, serglycin, in platelet exocytosis
Authors: Lykins Joshua , Chanzu Harry , Whiteheart Sidney
Published: June 2021

Platelet granule secretion is essential for the maintenance of the vascular system and influences cardiovascular diseases such as stroke and myocardial infarction. Platelets secrete their granules in response to vascular damage, which promotes clot formation; but, excessive secretion can lead to occlusive thrombosis. Platelets are derived from large progenitor cells called megakaryocytes that reside in the bone marrow. These cells project proplatelet extensions into circulation for platelet production. Platelet secretory granules are synthesized within megakaryocytes and packaged into proplatelets. Serglycin is an intra‐granular, chondroitin sulfate proteoglycan produced by several hematopoietic cells. Serglycin−/− mice exhibit decreased agonist‐dependent secretion of α‐granule cargo (Platelet Factor‐4 (PF4), MIP‐1) despite showing normal exposure of α‐granule membrane protein (P‐selectin), suggesting a defect in granule cargo storage and/or solubility. We hypothesize that the negative charges present on serglycin are vital for the retention and solubility of α‐granule cargo during their synthesis in megakaryocytes and packaging into platelet granules. We examined primary megakaryocytes isolated from bone marrow of wild‐type and serglycin−/− mice. Serglycin−/− megakaryocytes showed an increase in PF4 in the culture media, coupled with a decrease in PF4 associated with cell pellets, as measured by ELISA assays. Fluorescent microscopy also showed a decrease in the retention of fibrinogen, a soluble factor endocytosed by megakaryocytes and sorted into α‐granules, in serglycin−/− megakaryocytes. Additionally, myelofibrosis and splenomegaly were observed in serglycin−/− mice. These studies contribute to an understanding of the physiological function of serglycin in megakaryocytes and to the mechanism of α‐granule biosynthesis.

Support or Funding Information

Supported by HL56652, HL138179 and VA Merit Award

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inner mitochondrial translocase Tim50 is central in steroid metabolism in steroidogenic tissues
Authors: Bose Himangshu S , Marshall Brendan , Perry Elizabeth W , Whittal Randy M
Published: June 2021

Steroid hormones are synthesized in an acute condition but some genes are also transcriptionally active, and they are essential for survival of all mammals. The synthesis of metabolic reaction is initiated in the matrix side of mitochondria. In addition to the presence of different enzymes mitochondria has many translocases, which help in sorting and localization proteins across the subcompartments. The molecular basis of mitochondrial redistribution depends on the different steps of protein folding. In adrenal and gonads, Cytochrome P450 side chain cleavage enzyme (SCC or CYP11A1), catalyzes cholesterol to pregnenolone. We find that gradual mitochondrial import of SCC led to the association with mitochondrial translocase, Tim50. The N‐terminal is cleaved first followed by the C‐terminus amino acids, where deletion of more than ten amino acids from the C‐terminus reduced the activity and interaction with Tim50. Similarly, knocking down Tim50 ablates SCC activity and blocking complete SCC import into the inter mitochondrial membrane ablated activity. These two proteins are colocalized in the mitochondria, where amino acids 208–212 of Tim50 first interact with SCC. A mutation in either of these two proteins reduced their interaction, resulting in a metabolic and developmental disorder. Thus, the metabolism for conversion of pregnenolone depends on SCC and Tim50 together, as the negative feedback is inhibited in the absence of interaction between these two proteins, increasing ACTH levels.

Support or Funding Information

No

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Avocado Oil Ameliorates Non‐Alcoholic Fatty Liver Disease by Down‐Regulating Inflammatory Cytokines and Improving Mitochondrial Dynamics
Authors: Garcia‐Berumen Claudia Isabel , Olmos‐Orizaba Berenice Eridani , Márquez‐Ramírez Cristian Adrián , Orozco Alain Raimundo Rodríguez , González‐Cortez Aramen , Saavedra‐Molina Alfredo , Montoya‐Pérez Rocio , Cortés‐Rojo Christian
Published: June 2021

Nonalcoholic Fatty Liver Disease (NAFLD) is a chronic disease characterized by excessive fat accumulation, inflammation and liver dysfunction in the absence of significant alcohol consumption and without any other liver disease. NAFLD is accompanied by mitochondrial dysfunctions such as decreased activity of the enzymes of the electron transport chain (ETC), impaired β‐oxidation of fatty acids, excessive reactive oxygen species (ROS) production and increased lipid peroxidation. These last two processes have been involved in liver inflammation by augmenting the activity of cytokines like TNF‐α, and IL‐6. Mitochondrial dynamics, a process that modulates both mitochondrial morphology and function by events of fusion and fission, also becomes impaired during NAFLD by exacerbating mitochondria fission. Previously, we have reported that avocado oil, a rich source of C18:1, bioactive sterols and antioxidants, attenuates both mitochondrial dysfunctions and oxidative stress during diabetes and hypertension. Therefore, we aimed to test if avocado oil attenuates NAFLD by counteracting the alterations in both cytokines levels and mitochondrial dynamics in rats feed with a diet with high fat and fructose for 4 months. This diet led to hepatic alterations including inflammation, ballooning, necrosis and increased expression of both TNF‐α and IL‐6. Increased levels of ROS production and lipid peroxidation were observed in mitochondria, along with augmented expression of DRP1 and Fis1, two proteins of fission, and decreased levels of fusion proteins Mfn1/2 and OPA1. All these effects were counteracted when avocado oil was supplemented daily after one month of the beginning of the diet up to the end of the experiment. These data suggest that avocado oil ameliorates NAFLD by decreasing inflammation and improving mitochondrial dynamics. Thus, avocado oil may be a nutritional approach to complement pharmacological treatment of NAFLD.

Support or Funding Information

This work was supported by a Coordinación de la Investigación Científica‐UMSNH grant (to CCR)

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