bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ObjectiveOur 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.MethodsWe 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.ResultsUCP2 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.ConclusionOur 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 InformationNIH grants K08 HL135399 (SR); P01 HL114470 and R01 AG046210 (VJT), VA Merit Award I01BX003056 (VJT)
Schematic showing the role of UCP2 in fibroblast (patho)biologyimageSchematic showing the role of UCP2 in fibroblast (patho)biologyThis 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 InformationFunding 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 InformationThis 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 InformationNatural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant awarded to AWV; Alexander Graham Bell Scholarship (NSCERC) awarded to AAThis 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 InformationNIH5‐SC3‐GM116728This 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 InformationFunding: Purdue University MCMP Research Enhancement grantThis 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.