bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzyme Catalyzed Expressed Protein Ligation
Authors: Henager Samuel , Chu Nam , Chen Zan , Bolduc David , Wells James , Cole Philip
Published: November 2023

Here we show the utility of subtiligase, a previously described engineered form of the bacterial protease subtilisin that has been converted to a peptide ligase, for creating semisynthetic proteins from intein‐generated protein thioesters and synthetic peptides. Expressed protein ligation (EPL) is an established method that exploits a chemoselective (‘native chemical’) ligation between a recombinant protein thioester generated via an intein and an N‐Cys containing synthetic peptide. While a powerful method for protein semisynthesis, EPL is limited by its standard requirement for a Cys residue at the ligation junction and sometimes sluggish reaction rates. In this study, we investigate both ubiquitin and glutathione S‐transferase as model protein thioesters to assess the efficiency of subtiligase‐mediated protein ligation with respect to sequence selectivity proximal to the ligation junction. Many amino acid sequences at the junction were well tolerated and led to ligation conversions as high as 50–70% in 10 minutes or less. We also showed that subtiligase could be used to produce semisynthetic tetraphosphorylated PTEN, a lipid phosphatase and key tumor suppressor gene, in its fully native form. By using this semisynthetic tetraphosphorylated PTEN as a protein standard, we could more accurately quantify the level of tail phosphorylation of cell‐derived PTEN, allowing for insights into its regulation. The application of subtiligase described here therefore offers a rapid and efficient alternative to standard EPL for protein semisynthesis.

Support or Funding Information

NIH
MIF SUMOylation in regulation of its stability and activity during breast cancer progression and metastasis
Authors: Sen Progga , Zhang Xiang‐Dong
Published: November 2023

SUMOylation is an essential posttranslational modification that regulates many cellular processes ranging from cell cycle control to protein stability. Dysregulation of SUMOylation has been implicated in various diseases including cancers. Our recent studies revealed that SUMO‐2/3 modification of macrophage migration inhibitory factor (MIF) is greatly increased in metastatic breast cancer cells compared to non‐metastatic control cells. As a pro‐inflammatory and carcinogenic cytokine, MIF acts as a direct link between inflammation and cancer. We further showed that SUMO‐2/3 modification of MIF is positively correlated with its increased levels in metastatic cells compared to non‐metastatic cells, whereas inhibition of SUMOylation by overexpression of SUMO‐specific isopeptidase SENP1 or SENP2 greatly decreases levels of MIF. Furthermore, we demonstrated that MIF is SUMOylated at a single lysine residue, K78, using in vitro SUMOylation assays. MIF is known to be degraded through ubiquitin‐dependent proteolysis and is ubiquitinated at K78, suggesting that MIF SUMOylation can inhibit its ubiquitination and degradation. Lastly, our protein interaction assays have revealed that MIF specifically interacts with SUMO E3 ligase PIAS3. As both PIAS3 and MIF are often upregulated in various types of cancers including breast cancer, it would be very interesting to test whether PIAS3 is the E3 ligase for MIF SUMOylation and increases MIF stability. Hence, characterization of MIF SUMOylation is expected to enrich our understanding of the cross‐talk of SUMO and ubiquitin pathways during breast cancer progression and metastasis.

Support or Funding Information

Wayne State University Startup Grant

American Cancer Society Institutional Grant
Decreasing O‐GlcNAcylation of 8‐oxoguanosine DNA Glycosylase (OGG1) in Cardiac Myocytes Cultured in High Glucose Leads to Improvement of DNA Repair and Mitochondrial Function
Authors: Cividini Federico , Dai Anzhi , Scott Brian , Diemer Tanja , Suarez Jorge , Dillmann Wolfgang
Published: November 2023

DNA damage in cardiac myocytes resulting from increased oxidative stress is an important factor in the pathogenesis of diabetic cardiomyopathy. Although DNA repair machinery has been described in the cardiac myocyte, the regulation of oxidative DNA damage repair has been incompletely investigated. Moreover, in hyperglycemia the O‐GlcNAc post‐translational protein modification has been reported to interfere with the regular intracellular activity of metabolically relevant proteins in either an adaptive or a maladaptive manner. The enzyme 8‐oxoguanosine DNA glycosylase (OGG1) is key in DNA repair. Thus, the objective of this work was to investigate the influence of O‐GlcNAcylation of OGG1 in excessive glucose concentrations, simulating the levels observed in diabetic animals or humans. Neonatal cardiac myocytes (NCM) were isolated and cultured for 72 h in either normal glucose (NG; 5.5 mM glucose, 19.5 mM mannitol) or high glucose (HG; 25 mM glucose). The relationship between OGG1 O‐GlcNAcylation levels, enzymatic functionality and subcellular localization was analyzed. OGG1 was found to be up‐regulated and highly O‐GlcNAcylated in HG cultured NCM and was associated with increased 8‐OHdG (8‐hydroxy‐deoxyguanosine) levels, a common oxidative stress biomarker targeted by OGG1. In addition, decreased DNA quality and mitochondrial function were observed. Interestingly, OGG1 enzymatic activity in cell lysates was lower in HG compared to NG. Furthermore, in vitro experiments with recombinant OGG1 and O‐GlcNac transferase (OGT) demonstrated that O‐GlcNAcylation of OGG1 diminishes enzyme activity. We performed rescue experiments in NCM in HG by either rectifying OGG1 levels or reducing OGG1 O‐GlcNAcylation. NCM were either transduced with adenoviral particles (Adv) carrying OGG1 (Adv‐OGG1) or co‐transduced with Adv‐OGG1 and a dominant negative OGT (Adv‐dnOGT). Co‐transduced NCM showed improved OGG1 enzymatic activity compared to controls cells in spite of HG. These results were associated with lower 8‐OHdG levels. Finally, a different sub‐cellular enzyme distribution and an improvement in mitochondrial function were also detected. In conclusion, these results suggest that O‐GlcNAcylation of OGG1 could be considered a maladaptive phenomenon caused by hyperglycemia, which directly interferes with DNA repair by altering OGG1 activity and translocation, and indirectly leads to mitochondrial dysfunction.

Support or Funding Information

This work was supported by National Institutes of Health grants (5 P01 HL066941‐13), with a Merit Review Award from the Department of Veteran's Affairs (5 I01BX001121‐02) and the P. Robert Majumder Charitable Foundation.
Specificity of Protein Arginine Methyltransferases (PRMTs): How Does the Trypanosoma brucei PRMT7 Limit its Activity to Monomethylation of its Target Proteins?
Authors: Jain Kanishk , Warmack Rebeccah A , Feng You , Debler Erik W , Stavropoulos Peter , Blobel Gunter , Clarke Steven G
Published: November 2023

In the family of the nine mammalian enzymes that modify proteins by adding one or two methyl groups to the terminal guanidino nitrogen atoms of acceptor proteins, PRMT7 is unique in producing solely monomethylarginine (MMA) products. The other eight PRMTs catalyze the formation of symmetrically or asymmetrically dimethylated arginine (SDMA or ADMA) residues. Since these modifications are often recognized by reader proteins specific for MMA, SDMA, or ADMA, the biological functions of PRMTs are closely linked to the type of methylation. SDMA and ADMA residues on certain proteins have been known to up‐regulate or down‐regulate transcription, suggesting a possible regulatory function for MMA markers produced by PRMT7. In order to understand the nature of monomethylation and what keeps PRMT7 from dimethylating its substrates, we have focused our attention on the residues of its active site from the protozoan Trypanosoma brucei responsible for sleeping sickness in humans. By using site‐directed mutagenesis, we have mutated several residues including those in the “double E” loop known to interact with the methyl‐accepting substrate arginine residues and then assayed its level and type of methylation via high resolution cation exchange chromatography. Here we present results suggesting that most of these enzyme modifications result in significantly reduced activity but no change in MMA specificity. However, we have found that a mutation of glutamate‐181 (a double E residue) to an aspartate residue not only significantly diminishes enzyme activity but also confers to the enzyme the ability to asymmetrically dimethylate arginines, generating ADMA products. This result pinpoints a crucial residue in maintaining the specificity of PRMT7.

Support or Funding Information

EWD was a Frey Fellow of the Damon Runyon Cancer Research Foundation. This work was also supported by grants from NIH including GM026020 (S.G.C.) and a Ruth L. Kirschstein National Service Award GM007185 (to K.J. and R.A.W.).
5hmC Distribution in Isolated Visceral Adipose Tissue Nuclei
Authors: Yu Ping , Ji Lexiang , Lee Kevin , Yu Miao , He Chuan , Ambati Suresh , Schmitz Robert , Meagher Richard
Published: November 2023

The reprogramming of specific cell types, and in visceral adipocytes in particular, is likely to be a fundamental to obesity and its related comorbidities. We explored the hypothesis that adipose tissue contains epigenetically distinct subpopulations of adipocytes in that are differentially potentiated to record memories of their cellular environment. Adipocytes are large, fragile, and technically difficult to isolate. We developed fluorescence nuclear cytometry (FNC) and fluorescence activated nuclear sorting (FANS) of cellular nuclei from visceral adipose tissue (SsVAT) using the levels of the adipocyte‐specific protein, peroxisome proliferator‐activated receptor gamma‐2 (PPARg2). Using FANS we obtained highly purified PPARg2‐Positive (PPARg2‐Pos) nuclei, which were distinct from PPARg2‐Negative (PPARg2‐Neg) leukocyte, endothelial cell and progenitor cell nuclei. PPARg2‐Pos VAT nuclei showed 2‐ to 50‐fold higher levels of transcripts encoding most of the chromatin‐remodeling factors assayed, including those which regulate the methylation of histones and DNA cytosine (e.g., DNMT1, DNMT3A, TET2, TET3, KMT2C, SETDB1, PAXP1, ARID1A, KMT2C, JMJD6, CARM1, and PRMT5). PPARg2‐Pos nuclei have large decondensed chromatin structures. Immunofluorescence microscopy (IFM) revealed the co‐distribution of PPARg2 protein with 5‐hydroxymethylcytosine (5hmC), with nearly all nuclei staining most strongly for PPARg2 also staining strongly for 5hmC. TET‐assisted bisulfite sequencing (TAB‐seq) demonstrated 5hmCG levels were the highest for PPARg2‐High nuclei and lowest for PPARg2‐Neg nuclei across all gene regions, and 5hmC levels were remarkably dynamic in gene bodies of PPARg2‐Pos nuclei, dropping 3.8‐fold from the highest quintile of expressed genes to the lowest. In short, VAT‐derived adipocytes are epigenetically distinct with PPARg2 and 5hmC levels coordinately regulated and proportional to nuclear size.

Support or Funding Information

This work was supported by grants from UGA's Office of the Vice President for Research, UGA's Obesity Initiative and from NIH's National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, DK1003920 and DK096300) to RBM, NHGRI HG006827 grant to CH, the National Institutes of Health grant R00GM100000 to RJS, and a Patricia and Doyle Mote Biomedical Research Award to PY.
Overexpressed Gα13 stimulates Serum Response Factor mediated transcription concurrent with relocalization to a soluble fraction
Authors: McDiarmid Gray R , Tagliatela Alicia C , Meigs Thomas E.
Published: November 2023

Heterotrimeric G proteins consist of α, β, and γ subunits, and activating, GTPase‐deficient mutations in the α subunit of several classes have been implicated in tumor progression. The G12/13 class of α subunits, comprising Gα12 and Gα13, has the unique ability to stimulate oncogenic transformation of cultured cells though overexpression of the wildtype α subunit. However, the mechanism that allows this signaling property of wildtype Gα12 and Gα13 is not understood. Using transiently transfected HEK293 cells, we examined varying levels of constitutively active and wildtype forms of Gα13 for ability to stimulate serum response factor (SRF) mediated transcription, a cell growth signaling pathway implicated in melanoma and other cancers. Constitutively active Gα13 showed an approximate 100‐fold stimulation of SRF signaling, and unexpectedly, we observed a response fully half this magnitude using wildtype Gα13. In cell fractionation experiments using an epitope‐tagged wildtype Gα13, this transiently expressed protein began appearing in the soluble fraction as its levels increased, whereas endogenous Gα13 remained in the membrane‐associated fraction. Under conditions in which Gα13 redistribution was observed, SRF activation showed a sharp increase. This stimulatory effect of wildtype Gα13 was blunted by co‐expression of Gβ1 and Gγ2 subunits, suggesting overexpressed Gα13 drives SRF signaling due to stoichiometric imbalance in the heterotrimer. Conversely, SRF stimulation by wildtype Gα12 was enhanced by these Gβ/Gγ subunits. Gβ1/Gγ2 co‐expression also changed the subcellular distribution of wildtype Gα13 to a predominantly membrane‐associated state. Furthermore, mutation of two N‐terminal Cys residues required for Gα13 palmitoylation caused localization to the soluble fraction but abolished SRF signaling, suggesting this lipid modification as critical for the soluble, overexpressed pool of Gα13 to drive this growth pathway.

Support or Funding Information

We acknowledge support from the GlaxoSmithKline and C.D. Spangler Foundations, Lineberger Comprehensive Cancer Center, and UNC‐Asheville Undergraduate Research Program.
The Role of CaMKII in mGlu5 Signaling
Authors: Marks Christian Randal , Shonesy Brian C , Colbran Roger J
Published: November 2023

Ca2+/calmodulin (CaM)‐dependent protein kinase II (CaMKII) is a serine/threonine kinase that accounts for ~1% of total protein in forebrain. Interactions of activated CaMKII with CaMKII associated proteins (CaMKAPs) and roles of CaMKII in modulating ionotropic glutamate receptors and voltage gated ion channels have been studied intensively, but the role of CaMKII in G protein coupled receptor (GPCR) regulation is less well understood. Here, we confirmed a previous report1 that CaMKII interacts with the metabotropic glutamate receptor 5 (mGlu5), a Gq coupled receptor, in vitro, in HEK293 cells and in mouse forebrain lysates. Unlike most other CaMKAPs, mGlu5a interacts with INACTIVE CaMKII, and the interaction is disrupted by Ca2+/CaM. We also found that CaMKII phosphorylates the mGlu5a C‐terminal domain (CTD) in vitro. Inclusion of the mGlu5b‐specific splice insert in the CTD does not affect the binding or phosphorylation of mGlu5 by CaMKII. We have begun to characterize CaMKII binding and phosphorylation sites in mGlu5a/b. Binding of Ca2+/CaM to mGlu5 blocks the phosphorylation of some sites on mGlu5, such that they are preferentially phosphorylated by Thr286‐autophosphorylated CaMKII, which exhibits Ca2+/CaM‐independent kinase activity. In addition, initial studies indicate that the mGlu5 CTD modulates CaMKII activation in vitro, representing a potential bidirectional regulatory mechanism. We are currently comparing CaMKII activity and subcellular localization in wild‐type and mGlu5‐null mice. Understanding the interplay between these key synaptic regulators may provide mechanistic insights into their role in the modulation of synaptic transmission.

Support or Funding Information

T32‐DK007563 and AHA 15PRE25110020 to CRM

R01‐MH063232 and R01‐NS078291 to RJC
Screening for Potential Inhibitors of the Pro‐angiogenic GTPase TCL Using Split‐Venus Reporter Proteins
Authors: Florke Rebecca , Hamann Michael
Published: November 2023

TCL is a specific Rho GTPase involved in cytoskeletal signaling related to angiogenesis when it is activated and is implicated in the development of tumor vascularization. Therefore, TCL is a promising new target in anticancer treatments. In order to screen for inhibitors of TCL, a detection system was designed using the fluorescent protein Venus. The coding sequence for Venus was split to produce two non‐fluorescent fragments. TCL and the GTPase binding portion of PAK were then genetically fused to the two Venus sequences to detect GTP‐loaded TCL. Constitutively active (CA) and dominant negative (DN) mutants of TCL, as well as other Rho GTPases, were tested to establish the validity of this bimolecular fluorescence complementation (BiFC) method. In contrast to the DN GTPases, the CA GTPases yielded significantly higher fluorescence. To evaluate the ability of the BiFC assay to respond to inhibitors, a novel Cdc42‐specific inhibitor, ML141, was used with Cdc42 and resulted in decreased fluorescence. Initial screening of TCL with ML141 also produced a decrease in fluorescence, suggesting that ML141 may inhibit either the GTP loading of TCL or the interaction between GTP‐loaded TCL and PAK.
Immunofluorescence Localization of Ligand‐Induced Internalization, Trafficking, and Signaling of eGFP‐Tagged Guanylyl Cyclase/Natriuretic Peptide Receptor‐A into the Subcellular Compartments
Authors: Mani Indra , Tripathi Satyabha , Garg Renu , Pandey Kailash N
Published: November 2023

Cardiac hormone, atrial natriuretic peptide (ANP) activates guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), which produces second messenger cGMP and lowers blood pressure and blood volume. The objective of this study was to visualize the internalization and intracellular trafficking of NPRA in the subcellular compartments of intact cells. To study the internalization and intracellular trafficking of NPRA, we utilized immunofluorescence staining and co‐immunoprecipitation (co‐IP) of plasma membrane, endosomal, lysosomal, and Rab 11 markers to follow trafficking and signaling by confocal immunofluorescence microscopy and immunoblotting. A chimeric construct of enhanced green fluorescence protein (eGFP) and NPRA (eGFP‐NPRA) was used to study internalization and trafficking of receptor in human embryonic kidney‐293 (HEK‐293) cells. The treatment of cells with ANP at different times accelerated the internalization of receptor from cell surface to cell interior. Colocalization of eGFP‐NPRA with pan‐Cadherin indicated that internalization of receptor accounted for 58%, 69%, 70%, and 75% at 5, 10, 15, and 30 min, respectively, compared with untreated control cells. Colocalization of eGFP‐NPRA with early endosome antigen‐1 (EEA‐1) marker was maximum at 5 min (63%), and gradually decreased at 10 min (43%), 15 min (31%) and at 30 min (23%) respectively. Similarly, colocalization of NPRA with lysosome‐associated membrane protein‐1 (LAMP‐1) gradually increased at 5 min (13%), 10 min (36%), 15 min (42%), and at 30 min (44%). After treatment with lysosomotropic agents, intracellular accumulation of the receptor progressively increased at 5 min and continued up to 30 min. Rab 11 was used as a recycling endosome marker, which showed that 20% receptor recycled back on plasma membrane. Co‐immunoprecipitation assays confirmed that the localization of internalized receptors occurred with subcellular organelles during the endocytosis of NPRA. Immunofluorescence and enzyme‐linked immunosorbent assay (EIA) analyses showed a marked increase in the accumulation of intracellular cGMP concurrent with receptor internalization. Our study suggests that after ligand binding, receptor internalized and trafficked into subcellular compartments with concurrent generation of intracellular cGMP, which regulates blood pressure and cardiovascular homeostasis.

Support or Funding Information

This work was supported by NIH grants R01HL057531 and R01HL062147
Biological funtions for Ans‐linked oligosaccharides in the tethered rec‐eel luteinizing hormone
Authors: Byambaragchaa Munkhzaya , Min Kwan‐Sik , Kang Myung‐Hwa
Published: November 2023

Japanese eel Anguilla japonica is one of the most important fish species being aquaculture in Japan but is hampered from the fact that this speices does not reproduce in captivity. Artifical induction of gonadal maturation has been successful by administration of salmon pituitary extracts, but the understanding the regulatory mechanism of gonadal development moderated by FSH and LH remains elusive due to lack of suitable amounts of eel GTHs.

In the present study, we produced tethered rec‐eel LH and mutants (56, 79 and 56–79 of a‐subunit) of Asn‐linked oligosaccharides in CHO cells. In western blot analysis, its molecular weight was deteced at 32‐kDa in wild type. Elimination of the carbohydrate at a single site reduced with about 10%, where molelcular weight in double mutant decreased ove 20% compared to wild type. Now we are trying to analysis the biological activity and internalization using cells expressing eel LH/CG receptor cDNA.

Support or Funding Information

This work was supported by a scholarship form the BK21 Plus Program, the Ministry of Education, Science and Technology, Korea. This work was supported by a grant form KIMST (D11503115H480000110), Korea.
Exchange protein directly activated by cAMP (EPAC) regulates neuronal polarization through Rap1B
Authors: Schmidt Martina , Llancao Pablo Munoz , González‐Billault1 Christian
Published: November 2023

Acquisition of neuronal polarity is a complex process involving cellular and molecular events. The second messenger cyclic adenosine monophosphate (cAMP) is involved in axonal specification through activation of protein kinase A (PKA). However, an alternative cAMP‐dependent mechanism involves the exchange protein directly activated by cAMP (EPAC), which also responds to physiological changes in cAMP concentration, promoting activation of the small Rap GTPases. Here we present evidence that EPAC signaling contributes to axon specification and elongation. In primary rat hippocampal neurons, EPAC isoforms were differentially expressed during axon specification. Furthermore, 8‐pCPT, an EPAC pharmacological activation and genetic manipulations of EPAC in neurons induced supernumerary axons, indicative of Rap1b activation. Moreover, 8‐pCPT–treated neurons expressed ankyrin G and other markers of mature axons such as synaptophysin and axonal accumulation of vGLUT1. In contrast, pharmacological inhibition of EPAC delayed neuronal polarity. Genetic manipulations to inactivate EPAC1 using either shRNA or neurons derived from EPAC1 knock‐out mice lead to axon elongation and polarization defects. Interestingly, multiaxonic neurons generated by 8‐pCPT treatments in wild type neurons were not found in EPAC1 knock‐out mice neurons. Altogether these results propose that EPAC signaling is an alternative and complementary mechanism for cAMP‐dependent axon determination.
Novel role of bHLH protein Daughterless in synaptogenesis
Authors: Robinson Erin L , Waddell Edward A , D'Rozario Mitchell , Marenda Daniel R
Published: November 2023

Basic Helix Loop Helix (bHLH)proteins are a class of highly conserved transcription factors. Proneural bHLH proteins have been extensively studied for their role in neurogenesisregulation, however the role of class 1 bHLH proteins in postmitotic cells remains unknown. Class 1 bHLH proteins function by heterodimerizing with class II or V bHLH proteins, orhomodimerizing to alter transcription. Ourlab has identified a novel role of the class 1 bHLH protein, Daughterless (Da),in postmitotic neurons in Drosophila Melanogaster, in which it restricts synaptogenesis. Here, we show that Da performs this function in part through restricting Neurexin expression, a cell adhesion protein. Our data also suggests that Da cooperates with the class V bHLH protein, Extramacrochaetae (Emc), in postmitotic neurons. Emc is a well‐established binding partner of Da during development, where it serves to sequester Da, preventing DNA binding. Taken together, these results provide a novel role for the class 1 bHLH protein, Daughterless, in postmitotic neurons.
3D‐Printing and Casting of Biological Constructs for Generating Stem Cell‐Based Scaffolds
Authors: Alexander J. Steven , Wang Yuping , Ott Bryton , Al‐Kofahi Mahmoud , Kevil Chris G. , Mills David K
Published: November 2023

Although stem cells (SC) represent a powerful new approach for restituting organs and tissues injured by trauma or disease, their use largely anticipates the deliberate delivery or homing of these cells to target tissues and does not as often involve regenerating supportive structures which could represent tissue replacements. The creation of biocompatible 3D‐printed tissue designs, the use of matrix‐casting and the incorporation of programmatic molecular‐environmental cues may help move the field of SC tissue replacement towards ‘ready‐made/on‐demand’ originated from either patient SC or from specifically engineered SC. Vascular prostheses represent an important, novel and widely applicable use for stem‐cell scaffold generation. We are optimizing the use of printed resorbable polyvinyl alcohol supports with matrix casting to generate ‘vessels’ which when seeded with vascular–lineage cells mature over a period of days into replicant ‘vessels’ which progressively increase in tensile strength. The dimensions of these vessels can be ‘programmed’ in terms of its final length, diameter and wall thickness by varying scaffold composition and by varying the initial cell seeding densities in these constructs and by medium formulations. Perhaps most importantly, unlike many conventional cell printing technologies, the current approach does not require printing materials to be sterile until final stages of assembly permitting a wider spectrum of more complex designs.
Development of Biologically Inspired 3D Printed Biomaterial Scaffolds for Improved Stem Cell Differentiation
Authors: Zhang Lije Grace
Published: November 2023

As the most versatile and promising cell source, stem cells have been studied in regenerative medicine for a decade. However, despite their remarkable potential, one of the major challenges in successfully utilizing stem cells is the difficulty in providing proper environmental cues to regulate their self‐renewal and differentiation. While current available techniques utilizing a 2D microenvironment present easy and effective methods for investigating cell behavior in vitro, 2D culture systems fail to reflect cells’ native tissue environment. In native tissue, stem cells reside within a 3D extracellular matrix and are exposed to spatiotemporal chemical and physical cues, thus creating a biomimetic 3D tissue environment is a critical step for successfully controlling stem cell behavior. Our lab has developed advanced table‐top 3D bioprinting systems which can print 3D biomaterial constructs with precisely controlled microstructure and regional variation. Particularly, one significant component in our printing ink is biologically inspired nanobiomaterials, which can add further chemical signaling cues and mechanical strength to printed constructs. Our results show that these 3D printed scaffolds have not only improved mechanical properties but also can regulate human bone marrow mesenchymal stem cell growth and differentiation, thus promising for various tissue regeneration.
Hepatitis B virus X protein induces hepatic steatosis by enhancing the expression of liver fatty acid binding protein
Authors: Lin Xu , Wu Yun‐Li , Peng Xian‐E , Zhu Yi‐Bing , Yan Xiao‐Li , Chen Wan‐Nan
Published: November 2023

BACKGROUND & AIMS

Hepatitis B virus (HBV) has been implicated as a potential trigger of hepatic steatosis although the molecular mechanism involved in the pathogenesis of HBV‐associated hepatic steatosis still remains elusive. Our prior work has revealed that the expression level of liver fatty acid binding protein (FABP1), a key regulator of hepatic lipid metabolism, was elevated in HBV‐producing hepatoma cells. In this study, we sought to determine the effects of Hepatitis B X protein (HBx)‐mediated FABP1 regulation on hepatic steatosis and the underlying mechanism.

METHODS

mRNA and protein level of FABP1 were measured by quantitative RT‐PCR and western blot. HBx‐mediated FABP1 regulation was evaluated by luciferase assay, co‐immunoprecipitation and chromatin immunoprecipitation. Hepatic lipid accumulation was measured by Oil‐Red‐O staining and triglyceride level.

RESULTS

Expression of FABP1 was dramatically increased in HBV‐producing hepatoma cells, serum of HBV‐infected patients, and serum and liver tissues of HBV‐transgenic mice. Ectopic overexpression of HBx resulted in up‐regulation of FABP1 in HBx‐expressing hepatoma cells whereas HBx abolishment reduced FABP1 expression. Mechanistically, HBx activated FABP1 promoter in HNF3β, C/EBPα and PPARα‐dependent manner, in which HBx increased the expression of HNF3β and physically interacted with C/EBPα and PPARα. On the other way around, knockdown of FABP1 remarkably blocked lipid accumulation both in long‐chain fatty acids treated HBx‐expressing HepG2 cells and in a high fat diet fed HBx‐transgenic mice.

CONCLUSIONS

FABP1 is a key driver gene in HBx‐induced hepatic lipid accumulation via regulation of HNF3β, C/EBPα and PPARα. FABP1 may represent a novel target for treatment of HBV‐associated hepatic steatosis.

Support or Funding Information

This work was supported by the grants from National Natural Science Foundation of China (Grant number 81271822 and 81401657), State Key Project Specialized for Infectious Diseases (2012ZX10002002‐004‐006 and 2013ZX10002002‐005‐002) and Foundation of Fujian Educational Committee (Grant number JK2013021).
Dietary docosahexaenoic acid (DHA) halts progression of nonalcoholic steatohepatitis (NASH), but does not promote fibrosis remission in Ldlr−/− mice
Authors: Lytle Kelli A. , Wong Carmen , Jump Donald
Published: November 2023

Nonalcoholic fatty liver disease (NAFLD) is a major public health concern. NASH, the progressive form of NAFLD, is characterized by hepatosteatosis, inflammation, oxidative stress and fibrosis. NASH is a risk factor for cirrhosis, hepatocellular carcinoma and liver failure. We previously established that DHA attenuated the onset of western diet (WD)‐induced NASH in Ldlr−/− mice. Herein, we evaluate the efficacy of DHA to slow progression and promote disease remission in mice with pre‐existing NASH. Mice maintained on the WD for 22 weeks developed mild NASH. These mice were randomized to 3 groups: (Group 1) WD‐Base [euthanized at 22 weeks]; (Group 2) WD + Olive oil; and (Group 3) WD + DHA. Olive oil was added to ensure isocaloric diets and DHA was at a therapeutic dose, i.e., 2% of total calories. A Control Group was maintained on laboratory chow for the duration of the study. Mice in Groups 2 and 3 were euthanized after 8 weeks on the new diets. DHA had no effect on WD‐induced changes in body weight, food intake, plasma glucose, TLR4 agonists or leptin. DHA, however, decreased blood lipids (triglycerides, cholesterol) and Toll‐like receptor 2 agonists. Analysis of hepatic histology, lipids and gene expression established that the NASH phenotype worsened in WD + Olive oil group, but not WD + DHA. Hepatic gene expression markers of inflammation, oxidative stress and fibrosis in the WD‐Base and WD + DHA groups were comparable. While DHA supplementation increased hepatic omega‐3 fatty acids and repressed further total hepatic fatty acid accumulation, it failed to fully eliminate fibrosis. Taken together, these studies establish that DHA supplementation, at physiologically relevant levels, attenuates disease progression in mice with pre‐established NASH, but does not promote full NASH remission.

Support or Funding Information

Supported by NIH: DK094600
FTY720/Fingolimod Reduces Steatosis in a Murine Model of Non‐Alcoholic Fatty Liver Disease
Authors: Rohrbach Timothy D , Asgharpour Amon , Avni Dorit , Cazanave Sophie C , Bedossa Pierre , Sanyal Arun J , Spiegel Sarah D
Published: November 2023

Non‐alcoholic fatty liver disease (NAFLD) is a metabolic disease characterized by a spectrum of related liver disorders. Liver steatosis is an early initiator of NAFLD followed by the development of nonalcoholic steatohepatitis. Approximately 30% of adults in the United States are affected by NAFLD and although obesity and insulin resistance are risk factors for NAFLD, there is currently no approved therapy for this disease. To make matters worse, NAFLD can progress to liver cirrhosis and ultimately hepatocellular carcinoma. Sphingolipid metabolites, particularly ceramide and sphingosine‐1‐phosphate (S1P), have recently received attention for their potential roles in insulin resistance and hepatic steatosis. FTY720/Fingolimod, an FDA approved pro‐drug for treatment of multiple sclerosis, is phosphorylated in vivo to its active phosphorylated form by sphingosine kinase 2 and has been shown to interfere with the actions of S1P and inhibits several sphingolipid metabolic enzymes. Therefore, in this study we investigated its effect in the C57BL/6 x 129S murine model of NAFLD. Mice were fed a high fat diet (42% kcal from fat) with sugar water (HFD+SW) for 16 weeks with concurrent oral administration of a clinically relevant dose of FTY720 (0.3 mg/kg). FTY720 significantly decreased liver lipid accumulation and steatosis. Mass spectrometry analysis confirmed that FTY720 was phosphorylated to its active form, FTY720‐phosphate. Although levels of S1P and monohexosylceramide were significantly decreased in blood by administration of FTY720, no major changes in ceramide were detected. Moreover, liver sphingolipid levels including ceramide, monohexosylceramide, sphingomyelin, dihydroceramide, dihydromonohexosylceramide, and dihydrosphingomyelin were all decreased (p < 0.05) by FTY720 treatment. Additionally, FTY720 treatment caused a significant decrease in activation of Akt known to play a critical role in steatosis. Taken together, these findings support the notion that targeting sphingolipid metabolism could lead to reduced hepatic steatosis.

Support or Funding Information

Supported by NIH T32 Training Grant 5T32DK007150‐39.
Receptor for advanced glycation endproducts modulates leukotriene B4 receptor 1 signaling
Authors: Ichiki Takako , Koga Tomoaki , Okuno Toshiaki , Saeki Kazuko , Yamamoto Yasuhiko , Sakaguchi Masakiyo , Yokomizo Takehiko
Published: November 2023

Leukotriene B4 (LTB4) is a potent lipid chemoattractant for leukocytes. Leukotriene B4 receptor 1 (BLT1), a high‐affinity G protein‐coupled receptor (GPCR) for LTB4, is expressed in phagocytes and immune cells, and plays important roles in acute and chronic inflammatory responses. Although the LTB4‐BLT1 axis is known to promote inflammation, no studies have defined the binding proteins that modulate LTB4‐BLT1 signaling. Recent studies showed that receptor for advanced glycation end products (RAGE) modulates GPCR signaling via direct interaction with the receptors or downstream adapter molecules. RAGE is a member of the immunoglobulin superfamily, and is involved in several inflammatory disorders like BLT1. In this study, we investigated the functional interplay between BLT1 and RAGE. We found that RAGE and BLT1 were co‐immunoprecipitated and RAGE enhanced LTB4‐BLT1‐dependent ERK phosphorylation. RAGE inhibited LTB4‐BLT1‐dependent activation of nuclear factor‐κ B (NF‐κ B) and up‐regulation of pro‐inflammatory cytokines and chemokines. RAGE‐dependent inhibition of NF‐κ B was blunted by treatment with a MEK inhibitor, suggesting that RAGE suppresses LTB4‐BLT1‐dependent NF‐κ B signaling by enhancing MEK‐ERK pathway. Meanwhile, the velocity of LTB4‐dependent neutrophil migration was attenuated by soluble RAGE, which acts as an inhibitory decoy protein for RAGE signaling, or by RAGE deficiency. Furthermore, both LTB4‐dependent ERK phosphorylation in neutrophils and LTB4‐dependent chemotaxis in a murine peritonitis model were attenuated in RAGE‐deficient mice compared with wild‐type mice. These results indicate that RAGE potentiates LTB4‐dependent neutrophil migration by enhancing ERK phosphorylation. Collectively, our findings reveal that RAGE interacts with BLT1 and modulates LTB4‐BLT1 signaling through the potentiation of the MEK‐ERK pathway.

Support or Funding Information

T. Ichiki is a Research Fellow of the Japan Society for the Promotion of Science. This work was supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Nos. 22116001, 22116002, 15H05901, 15H05904, 15H04708, 25860223, 24590386, 15K08316, 24102522 and 25460374).
Biochemical Characterization of HDL/LDL‐like Particles Secreted by Astrocytes
Authors: Nguyen Amanda , Cabrera Karina
Published: November 2023

Lipids from the diet or those synthesized in the liver are packaged with proteins into HDL, LDL, and VLDL particles that circulate to places where they are needed. These particles are lipid core vesicles with protein wrapped around the external surface. However, these particles can not cross the blood brain barrier due to their size, so the bulk of neural lipid must be synthesized de novo in the brain. With their extensive processes of dendrites and axons, neurons have a plasma membrane surface several hundred times larger relative to a cell of the same soma size in another tissue and thus have much more lipid to synthesize, manage, and keep healthy. In order to address this biosynthetic need, the brain uses astrocytes to synthesize the bulk of the lipid and then transport it to neurons. Lipid and cholesterol synthesized in astrocytes are packaged into “HDL/LDL‐like” particles along with apolipoproteins and are released to be picked up by adjacent neurons with special receptors that recognize the apolipoproteins on the surface of these particles. Our initial proteomic studies identified ApoE and Apo1A as major components of the astrocyte secretome along with ApoC. Utilizing astrocytes isolated from mice at different ages from 4 to 28 months, we observed that ApoE protein levels secreted from the astrocytes decreased 3.5 fold in the oldest animals. We have hypothesized that the reduction in apolipoprotein production, ApoE in particular, is associated with a decrease in the maintenance of the extensive neuronal plasma membrane which results in enhanced susceptibility of the aged animal for damage to neurons and subsequent degeneration. We have developed an equilibrium sucrose centrifugation protocol to isolate these HDL/LDL‐like particles based on density as well as a more rapid isolation procedure using Cleanascite media to isolate all the lipid containing material and apolipoproteins from the astrocyte conditioned media. We are currently performing quantitative GC‐MS analysis to determine the lipid composition of these particles and if that composition changes with the age of the astrocytes. Western blot analysis is being utilized to determine if the apolipoprotein composition also changes with age of the astrocytes. These studies will clarify the role of lipid turnover/maintenance in the brain as it relates to the progression of neurodegenerative processes.

Support or Funding Information

McNair Scholars Program, Cowles Endowed Professorship Research Fund, Trinity University
Characterization of a Glycerophosphocholine Acyltransferase (GPCAT) from Saccharomyces cerevisiae
Authors: Anaokar Sanket , Lager Ida , Glab Bartosz , Banas Antoni , Stymne Sten , Patton‐Vogt Jana
Published: November 2023

Glycerophosphopholipids (phospholipids) are essential components of biological membranes. The maintenance of membrane composition through regulated synthesis and turnover of phospholipids is crucial to cell functioning. Phosphatidylcholine (PC) is the major phospholipid found in eukaryotic membranes. The two main pathways by which PC is synthesized in yeast are the methylation pathway and the Kennedy pathway (CDP‐DAG pathway). However, a novel pathway has been identified in S. cerevisiae and plants. This pathway involves glycerophosphocholine (GPC), the product of PC deacylation, as the starting substrate. It is proposed that GPC is converted to lysophosphatidylcholine (lysoPC) by accepting an acyl chain. This conversion is catalyzed by an enzymatic activity termed glycerophosphocholine acyltransferase (GPCAT). The lysoPC is then acylated, primarily by a lysophosphatidylcholine acyltranferase (LPCAT) encoded by ALE1, to form PC. Here we report on the identification of the gene encoding GPCAT and the characterization of this pathway in S. cerevisiae. The yeast deletion set was screened to identify the GPCAT‐encoding gene. Biochemical studies utilizing microsomes derived from strains lacking or overexpressing GPCAT in wild type and ale1 delete strains confirm in vitro GPCAT activity. In vivo metabolic labeling studies employing radiolabelled GPC were performed in various strain backgrounds in which the established pathways for PC biosynthesis were compromised and the GPCAT gene was either missing or overexpressed. Overall, our results indicate that the GPCAT gene acts as an GPC acyltransferase and that this novel pathway contributes to PC biosynthesis in the cell.

Support or Funding Information

National Institute of Health Grant: R15 GM104876
Phosphorylation of yeast glycerol 3‐phosphate acyltransferase Gpt2 regulates the timing of TAG synthesis and lipid droplet morphology
Authors: Tavassoli Marjan , Nagler Bernadette , Shabits Brittney , Sipila Patrick , Athenstaedt Karin , Zaremberg Vanina
Published: November 2023

Glycerol‐3‐phosphate acyltransferases catalyze the acylation of glycerol‐3‐phosphate at the sn‐1 position yielding 1‐acyl glycerol‐3‐phosphate (lyso‐phosphatidic acid). This is the first and rate limiting reaction in de‐novo synthesis of phosphatidic acid (PA), the key intermediate for the formation of all glycerophospholipids and triacylglycerols. In all eukaryotic organisms glycerol‐3‐phosphate acyltransferases as well as acyltransferases catalyzing the subsequent acylation reaction converting lyso‐PA to PA occur in redundancy. This led to the hypothesis that isoenzymes are forming different PA pools which supply different pathways. In the model organism yeast Saccharomyces cerevisiae two glycerol‐3‐phosphate acyltransferases have been identified, Gpt2 (Gat1) and Sct1 (Gat2). Most recently we demonstrated the important role of Gpt2 (but not Sct1) in cells challenged by oleic acid as the only carbon source. Whereas growth in oleic acid containing medium induced massive lipid particle accumulation in wild‐type cells, cells lacking GPT2 failed to accumulate these storage compartments of triacylglycerols and other non‐polar lipids, exhibited a strong growth defect and cell viability was significantly reduced. We were intrigued by the fact that the intracellular distribution of Gpt2 and its phosphorylation status changed under these growth condition compared to growth on glucose containing medium. We have identified several phosphorylation sites in Gpt2 by mass spectrometry, which are localized to a serine rich region in the carboxy‐terminus of the protein. Serine to alanine or to aspartate mutations as well as deletion of the serine rich region containing these residues, were introduced in the yeast genome to investigate the role of phosphorylation in the regulation of Gpt2. Importantly, all phosphorylation mutants were able to support life of cells lacking Sct1, indicating these mutants are functional. We show that manipulations of Gpt2 phosphorylation status strongly and specifically affect TAG accumulation, but not metabolism of the most abundant glycerophospholipids during late log phase of growth. Importantly, alterations in the TAG content are accompanied by marked changes in lipid droplet morphology in the respective mutants. Altogether our results support a role for Gpt2 phosphorylation in regulating the timing of TAG synthesis and lipid droplet morphology.

Support or Funding Information

This work has been financially supported by the Natural Sciences and Engineering Research Council of Canada (to VZ and BS) and the Austrian Science Fund (FWF; Project P21251 to KA).
Molecular biology of calcium trigger vesicle fusion in situ
Authors: Cialowicz Katarzyna , Duncan Rory , Rickman Colin , Brown Euan , Euston Matthew
Published: November 2023

Calcium ions in the human body serve as second messengers and are responsible for cell homeostasis. Muscle contractions and neuronal communication are supported by electrical activity. Voltage‐activated calcium channels are crucial for many cellular events. They play an important role in transducing the initial stimulus to the effector systems that modulate insulin secretion in pancreatic cells or neurotransmitter at nerve terminals. Dysfunction of these processes may cause many conditions, including diabetes or Alzheimer's disease. In recent years the amount of research on Ca2+ channels has markedly increased, but there are limitations related to physical restrictions in the spatial resolution of fluorescence microscopy. This phenomenon results in a loss of information with regard to the true location of a point source that is emitting light. Newly developed methods such as Stimulated Emission Depletion Microscopy (STED) or Photoactivated localization microscopy (PALM) allow imaging close to the molecular scale.

The general aims of this project so far were focused on understanding the mechanism of action and distribution of N‐type calcium channels by using novel tools I have helped to develop for calcium channel imaging.

To find out if Cav2.2 Ca2+ (Neuronal type) channels are in a clustered conformation or randomly distributed in the cellular plasma membrane, gated‐ (g)STED was used. After measuring the size of the signals in gSTED image data we conclude that Cav2.2 are patterned in clusters at the plasma membrane. To support that conclusion and characterize our imaging capability, more experiments using known size, sub‐diffraction fluorescent beads were conducted.

To quantify the mobility and location of N‐type calcium channels PALM was used. The principle of PALM is activating several photoactivable fluorescent molecules and imaging their signals until irreversible photo‐bleaching occurs. This process of activation and inactivation is then repeated over thousands of frames to ensure all molecules have been imaged. Using a photo‐uncageable fluorescent dye mono‐conjugated with a peptide toxin from a marine snail, allows for imaging single endogenous channel nano‐scale locations. This experiment is significantly better than the current state‐of‐the‐art and revealed the patterning of large cohorts of single endogenous ion channels. To verify mobility of N‐type calcium channels series of images were analysed using bespoke particle tracking scripts.

Exocytosis is a process in which secretory vesicles are fused to the plasma membrane. Vesicles proximal to the plasma membrane are triggered to fuse by a rise in local free Ca2+ ions. The probability of the release of synaptic vesicles is hypothesized to increase with the number of proximal calcium channels. We combined our novel approaches to generate calcium ‘activity maps’ of the plasma membrane to investigate the relationship between calcium channels and exocytosis.