bluuebunny/crossref_metadata_2025_split huggingface dataset:
PREFERENTIAL DIMETHYLATION OF HISTONE H4‐LYSINE 20 BY SUV4‐20
Authors: Yang Hongbo , Pesavento James J. , Wallrath Lori L. , Kelleher Neil L. , Mizzen Craig A. 
Published: June 2021

Mounting evidence suggests mono‐ (1m), di‐ (2m) and trimethylation (3m) of histone H4 at lysine 20 (K20) in higher eukaryotes are functionally distinct, involving in gene activation, DNA damage responses, and heterochromatin function, respectively. The SET domain proteins PR‐Set7 and Suv4‐20 have been implicated in forming 1mK20‐H4 and 3mK20‐H4, respectively, but enzymes for 2mK20‐H4 have not been unequivocally identified. Using RNAi targeting SET domain proteins repertoire in Drosophila in conjunction with Top Down mass spectrometry in a systematic screen for K20 methyltransferases, we discovered Suv4‐20 preferentially forms 2mK20‐H4 accounting for more than 80 % of all H4 in addition to forming 3mK20‐H4 comprising less than 5 % of all H4 in human and Drosophila cells. HP1 is required for normal levels of 3mK20‐H4, but not 2mK20‐H4, suggesting HP1 regulates Suv4‐20 product specificity. Marked alterations in K20 methylation levels due to Suv4‐20 depletion did not affect H4‐K16 acetylation, revealing these modifications are not competitive in vivo. Our data implies Suv4‐20 proteins have at least two distinct functions: directing dimethylation throughout the genome to facilitate DNA damage responses or other processes and directing rarer trimethylation to contribute to telomere function or other processes localized at heterochromatin.Work is supported by Roy J. Carver Charitable Trust (grant 04‐76).
SPFH1 and SPFH2 form a high molecular weight complex in the ER membrane that mediates the ER‐associated degradation of inositol 1,4,5‐trisphosphate receptors and other substrates in mammalian cells
Authors: Pearce Margaret M.P. , Wang Yuan , Wilkens Stephan , Wojcikiewicz Richard J.H. 
Published: June 2021

The endoplasmic reticulum (ER)‐associated degradation (ERAD) pathway is responsible for the degradation of aberrant proteins in the ER and the regulated degradation of several ER resident proteins. Inositol 1,4,5‐trisphosphate receptors (IP3Rs) form tetrameric Ca2+ channels in the ER membrane and are converted from stable proteins into ERAD substrates following their activation by IP3 and Ca2+. Using activated, endogenous IP3Rs as a tool, we have recently identified two novel mediators of the mammalian ERAD pathway—SPFH1 and SPFH2 (SPFH domain‐containing protein, members 1 and 2). SPFH1 and SPFH2 associate with IP3Rs immediately after their activation but prior to their polyubiquitination, and depletion of either SPFH1 or SPFH2 by RNAi inhibited the ERAD of IP3Rs and other mammalian ERAD substrates. SPFH1 and SPFH2, both ER membrane proteins that protrude into the ER lumen, were found to exist exclusively as an ∼1MDa complex by native PAGE, and TEM of purified, endogenous SPFH1/2 complex indicates that it assembles into ring‐like structures. These results suggest that SPFH1 and SPFH2 form a high molecular weight, ring‐shaped complex in the ER membrane that plays a key role in the early steps of mammalian ERAD pathway, perhaps as a factor involved in substrate recognition or retrotranslocation.Sources of funding: NIH, PhRMA Foundation
Novel supercomplexes of the proteasome in regulation of protein biosynthesis
Authors: Acs Klara , Perreard Laurent , Amir Omar , Groisman Regina , Gerber Scott , Kisselev Alexei 
Published: June 2021

26S proteasomes are large (1.6–2.5 MDa) multi‐functional proteolytic complexes that degrade the majority of proteins in mammalian cells. Proteasome inhibitors selectively induce apoptosis of malignant cells; one compound of this class, Velcade, is being used for treatment of multiple myeloma and is in clinical trials for the treatment of other malignancies. We have recently discovered that the majority of proteasomes in mammalian cells exist in much larger complexes. We have purified these supercomplexes and analyze their composition by mass spectrometry. This proteomic analysis has identified ribosomes and multi‐subunit translation‐initiation factor eIF3, and several RNA‐binding proteins, as principal components of these complexes, along with proteasomes. Hence, we hypothesize that proteasomes play a role in the initiation of translation, most likely by destroying translation inhibitor proteins and/or RNA‐binding proteins that protect mRNA from degradation by nucleases but also act as translational repressors. We introduce in this work the proteasome involvement in translation initiation and the interaction of proteasome‐eIF3 supercomplexes. These findings reveal a novel and unexpected link between protein synthesis and degradation and indicate that connection between these processes is tighter than previously thought.This work was supported by NCCC 203959 to AK.
Mature dendritic cells express functional thrombin receptors triggering chemotaxis and CCL18/PARC chemokine induction
Authors: Syrovets Tatiana , Li Xuehua , Paskas Svetlana , Laumonnier Yves , Simmet Thomas 
Published: June 2021

Monocyte‐derived immature dendritic cells (DC) do not express protease‐activated receptors (PARs). However, upon maturation with LPS, but not with TNF‐α or CD40L, DC express PAR1 and PAR3, but not PAR2 or PAR4. Stimulation of matured DC with thrombin, PAR1‐ or PAR3‐activating peptides elicits actin polymerization and concentration‐dependent chemotactic responses in LPS‐, but not in TNF‐α‐matured DC. Stimulation of PARs with thrombin or the respective receptor‐activating peptide activates ERK1/2 and Rho kinase 1 (ROCK1) as well as subsequent phosphorylation of the regulatory myosin light chain 2 (MLC2). The ERK1/2‐ and ROCK1‐mediated phosphorylation of MLC2 was indispensable for the PAR‐mediated chemotaxis as shown by pharmacological inhibitors. Additionally, thrombin stimulated the Rho‐dependent release of the CC chemokine CCL18/PARC, which induces chemotaxis of lymphocytes and immature DC as well as fibroblast proliferation. The colocalization of CD83+ DC with CCL18 in human atherosclerotic plaques as revealed by immunofluorescence microscopy combined with the presence of functionally active thrombin receptors on mature DC point to a previously unrecognized functional role of thrombin and its receptors in DC biology.This work was supported by the Deutsche Forschungsgemeinschaft.
Neutrophil elastase cleaves VEGF to generate a VEGF fragment with altered activity
Authors: Kurtagic Elma , Nugent Matthew A 
Published: June 2021

Vascular Endothelial Growth Factor (VEGF) and neutrophil elastase (NE) are implicated in the progression of emphysema. We investigated the potential connection between VEGF and NE injury. 125I‐VEGF was treated with NE, and the product visualized by SDS‐PAGE. We found that NE cleaves VEGF165 to a ∼34kDa disulfide linked fragment. VEGF interacts with cell surface receptors and components of the extracellular matrix, such as fibronectin and heparan sulfate. Hence, we examined the binding of the NE generated VEGF fragment (VEGFf) to VEGFR(1 and 2)‐Fc constructs, and found that VEGFf retained the ability to bind VEGFR1 but lost the ability to bind VEGFR2. We also examined VEGFf binding to fibronectin ± heparin and found that, unlike intact VEGF165, VEGFf did not bind fibronectin. VEGFf bound heparin, albeit with a reduced affinity compared to intact VEGF165. We assessed the activity of VEGFf in endothelial cells, and found that it did not activate ERK1/2 alone yet it enhanced VEGF165 activation of ERK1/2. We performed migration assays using macrophage RAW264.7 cells that express VEGFR1, and found that VEGFf and VEGF stimulated migration to a similar extent. VEGFf also activated Akt in endothelial and RAW264.7 cells. Our data indicate that NE generated VEGFf has altered activity, which might lead to functional changes within the injured lung.Supported by HL56200 and HL46920.
ATP hydrolysis in rho: identifying the residues and their roles
Authors: Balasubramanian Krithika , Stitt Barbara 
Published: June 2021

Escherichia coli transcription termination factor rho is an RNA/DNA helicase that terminates transcription using energy derived from the hydrolysis of ATP. The ATP binding sites of rho are located at the interfaces of adjoining C‐terminal domains and have the Walker A and B motifs, characteristic of many ATPases (Skordalakes & Berger, 2003; Richardson 2002).Available rho crystal structures capture the protein with its active site in an open configuration that must close to permit ATP hydrolysis. Because of this, the identity of active site residues predicted to mediate ATP hydrolysis is uncertain. To determine which amino acids activate water, stabilize transition state, sense the γ‐phosphoryl group, and coordinate the magnesium ion of MgATP, we are carrying out site‐specific mutagenesis on candidate residues which are conserved across bacterial species, and characterizing the relevant properties of the mutant proteins.Support: Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health
Probing the Mechanism of Cysteine Dioxygenase through Substitution of Conserved Active Site Residues
Authors: Imsand Erin , Ellis Holly R 
Published: June 2021

Cysteine dioxygenase (CDO) catalyzes the oxidation of L‐cysteine to L‐cysteine sulfinic acid. This reaction is the rate‐limiting step in the eukaryotic taurine biosynthetic pathway, and taurine levels are directly correlated to cysteine dioxygenase activity. Taurine is an important antioxidant and low levels of taurine have been linked to a variety of cardiac, neural, and autoimmune disorders.Catalytic mechanisms for CDO have been proposed based on recently published three‐dimensional crystal structures, although little direct spectroscopic or kinetic evidence is available to verify these proposals. To probe the mechanism, site‐directed mutagenesis was performed on conserved residues located in the active site of CDO adjacent to the iron center. Substitutions were made for Tyr58, Arg60, Ser153, and His155, replacing each residue with Ala. Kinetic parameters were established and compared to wild‐type to determine if any of the substitutions plays a role in catalysis. Several spectroscopic probes were used to characterize the metal coordination environment. Electron paramagnetic resonance (EPR) spectroscopy in combination with nitric oxide binding studies were performed to simulate the binding of dioxygen with or without the cysteine substrate. Amino acid residues critical to catalysis have been identified in these studies, and a mechanism for cysteine oxidation by CDO is proposed.
Defining the Role of Active Site Residues in the Activity of Glyoxysomal Malate Dehydrogenase
Authors: Jean Sophonie , Bell Ellis 
Published: June 2021

Although the structure of Malate Dehydrogenase has long been known and a mechanism proposed involving a his‐asp pair in the active site, little is known about the roles that residues further from the active site may play. Analysis of the active site cavity, both visually and by QM‐MM approaches, led to the construction of active site mutants and second sphere mutants [involving residues located 5–10A from the site of catalysis]. Using site directed mutagenesis, kinetics studies and x ray crystallography the roles of both residues directly in the active site and those further afield have been examined. H220 and D193 play well characterized roles in proton abstraction, however the pH dependence of the D193N mutant suggest other factors may be involved. Second sphere residues, N124, T152, T186, A183, G180, V164, N165, S166 and H90 have been mutated, expressed and studied by kinetics and stability studies. N165D has similar Vmax values at pH 7 & 8 but a marked increase in substrate inhibition at pH 7 and a reversal of the pH effects on Km relative to the native protein or S166A. The N165D and the S166A mutants have been crystallized and 3D structure determination is in progress. It is clear that a number of residues within this second sphere contribute to protein stability and aid in the fine tuning of the regulatory effects of substrate inhibition and pH Dependence.Supported by NF Grant MCB 0448905 to Ellis Bell
Laser Capture Microdissection: Combining LCM with IHC to investigate NMDA receptor subunit composition
Authors: Curry John Aloysius , Brinkmann Hannah , Richard Virgile , Rigby Mike 
Published: June 2021

The Neurokinin 1 receptor is a member of the tachykinin receptor sub‐family. It is distributed throughout the CNS, although to a lesser extent than its natural agonist, Substance P.There is much evidence to implicate the NK1 receptor within the physiology of acute pain, such as the anatomical localisation of Substance P and NK1 receptors within lamina I of the spinal cord, as well as correlative increases in Substance P production and NK1 receptor internalisation in response to increasing noxious stimuli. Reproduction of in vivo efficacy in the clinic has, however, been unsuccessful.Selective NR2 subunit mediated NMDA antagonism has also shown in vivo efficacy, expression of this subunit within the superficial lamina of the spinal cord adding anatomical evidence to support this mechanism. However, co‐expression of the NK1 receptor and the NMDA receptor has not been reported.We have developed a methodology to allow the isolation of high quality RNA, via Laser Capture Microdissection, following the immunolabelling of specific proteins of interest.Using this technology, we validated methodology via PCR of dissected samples, as well as looking at differential expression of the NR2A and NR2B receptor subunits on NK1 +ve and NK1 –ve samples taken from the rat striatum.
Sodium butyrate induction of apoptosis in human fibroblasts in culture
Authors: Henry Amanda J , Wagner Rebecca l , Birckbichler Paul J 
Published: June 2021

Tissue transglutaminase (tTg) is a multifunctional enzyme possessing two catalytic sites. One is a calcium‐dependent reaction that leads to formation of isodipeptide cross‐linkages. The cross‐linkages lead to the formation of a protein scaffold in cells undergoing apoptosis. GTP, a negative regulator for this cross‐linking activity, shifts the enzyme's catalytic activity to G‐protein activity.Human embryonic fibroblast cells (WI‐38) and their simian virus‐transformed counterparts (VA13A) grow adherent in vitro. Quantification of the amount of tTg using a fibronectin binding assay showed that VA13A cells contained 0.04493 ng tTg/uL sample in the supernatant and a non‐detectable amount in the pellet fraction. This amount increased to 8.2622ng tTg/uL sample in the supernatant fraction after the VA13A cells were exposed to sodium butyrate. These results were confirmed by indirect immunofluorescence microscopy, electrophoresis and Western blotting.Sodium butyrate induced apoptosis in VA13A cultures, but had no effect on apoptosis in WI‐38 cultures. Preliminary studies with sodium butyrate alone and in combination with calcium ionophore showed not only an increase in apoptosis but also an increased transglutaminase expression. Additionally, sodium butyrate treatment induced growth arrest in both cell lines and reverted the VA13A cells to a more “normal” morphology. These results taken collectively are indicative of a dual role of sodium butyrate; one involving growth arrest and the other apoptosis.
Microvillar protein trafficking and dynamics imaged by TIRF microscopy in living cells
Authors: Breusegem Sophia Y , Blaine Judith T , Doctor R Brian , Barry Nicholas P , Levi Moshe 
Published: June 2021

Polarized epithelial cells have distinct membrane domains. The apical domain consists of microvilli, is separated from the basolateral domain by tight junctions, and contains transporters and ion channels that are regulated by endo‐ and exocytosis from and to the intervillar cleft. Total internal reflection fluorescence (TIRF) microscopy is ideally suited to study endo‐ and exocytosis at the basolateral surface of cells grown on coverslips; however, in this set‐up it can not be used to study apical trafficking as the TIRF excitation field will not reach the apical membrane. We have developed a TIRF method to follow apical endo‐ and exocytosis, growing cells on membrane supports and turning them over onto coverslips. We show that apical TIRF does not lead to de‐differentiation and can be used to follow microvillar trafficking processes and dynamics in living cells with high spatial and temporal resolution. We use this method to study endocytosis of renal sodium/phosphate co‐transporters (NaPi‐2a and NaPi‐2c) in response to parathyroid hormone or high phosphate. We show that microvillar actin turnover occurs on the same time scale as NaPi‐2a internalization. In addition, we show trafficking of Shank2E, a PDZ‐domain containing NaPi‐2a‐binding protein, in the microvilli.S.Y.B. is supported by a postdoctoral fellowship from the American Heart Association, J.T.B. by a NRSA fellowship, and N.P.B. and M.L. by NIH.
Type‐I interferons inhibit Delta‐like‐1‐dependent T cell development and increase apoptosis of developing thymocytes in vitro
Authors: La Motte‐Mohs Ross , Gauchat‐Feiss Dominique , Baron Marie‐Laurence , Démoulins Thomas , Awong Génève , Michiels Thomas , Perreault Claude , Zúñiga‐Pflücker Juan Carlos , Sékaly Rafick‐Pierre 
Published: June 2021

Individuals with HIV/AIDS exhibit a gradual depletion of CD4+ T cells in the periphery and a chronic loss of T cells within the thymus. Strikingly, the thymus from these individuals exhibits increased levels of type‐I interferons (IFNs): α(s) and β, suggesting that developing thymocytes are susceptible to apoptosis through increased production of type‐I IFNs. To determine whether type‐I IFNs directly effect T cell development, we cultured fetal liver‐derived mouse hematopoietic stem cells (HSCs) on OP9‐DL1 cells and examined through flow cytometry the CD4−CD8− double negative (DN) and CD4+CD8+ double positive (DP) T cell populations that emerged. Specifically, addition of type‐1 IFNs reduced cellularity and blocked T cell differentiation at the DN1 to DN2 transition in a dose‐dependent manner. Type‐I IFNs mediated their effects directly to the HSCs independent of the OP9‐DL1 cells, as HSCs obtained from IFN‐receptor deficient (IFNAR−/−) mice, but not IFN‐receptor sufficient (IFNAR+/+) mice, were refractive to IFN treatment, exhibited increased cellularity, and progressed to the DP stage. Increased apoptosis over time was observed in IFN‐susceptible, but not IFN‐insensitive fetal liver cells. These in vitro studies have also been extended to the development of human T cells from cord blood‐derived HSCs.Supported by grants from the OHTN and CIHR.
Tamalin may cooperate with Cybr in modulating leukocyte migration in vivo
Authors: Coppola Vincenzo , Tessarollo Lino 
Published: June 2021

Cybr (aka pscdbp, CASP or CBP) is a scaffold protein that binds cythohesin‐1, a guanine nucleotide exchange factor for the ARF‐GTPases, which affects the actin cytoskeleton remodeling during cell migration. Indeed, we have shown in mouse that Cybr deficiency negatively affects the number of leukocytes circulating in blood and lymphocytes migrating to the lymph nodes especially in response to pro‐inflammatory cytokines in stress conditions. Notably, Cybr‐deficient mice show clear deficits of CTL‐mediated virus‐induced tumor rejection. Cybr is not expressed in the nervous system where its homolog Tamalin (aka Grasp) is expressed the most. Tamalin too is a scaffold protein and shares with Cybr high homology at the PDZ and coiled‐coil domain suggesting common functions. Initially, Tamalin has been described as absent from the immune system. We show that Tamalin, although at low levels compared to Cybr, is expressed in the immune system. Tamalin is co‐expressed with Cybr in specific cell types suggesting a non‐redundant role for these two closely related proteins.The goal of this study is to investigate in vivo whether Tamalin deficiency affects leukocyte migration and augment defects due to Cybr deficiency. For this purpose, we have generated Tamalin deficient mice and subsequently Cybr/Tamalin double knock‐out mice. We have previously shown that Cybr has a role in cell migration in stress conditions. Here, we show that Tamalin defcient mice do not show any obvious deficit of leukocyte migration. However, preliminary results suggest that Tamalin deficiency may strengthen Cybr migration defect in specific conditions. Further investigation is needed to clarify how these two proteins modulate cell migration in vivo. This research is supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
CD38 AND ZAP‐70 REGULATE CXCL12‐MEDIATED CHEMOTAXIS OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) CELLS
Authors: Deaglio Silvia , Vaisitti T. , Aydin S. , Bergui L. , D'Arena G. , Bonello L. , Omede' P. , Malavasi F. 
Published: June 2021

Absence of mutations in the IgV genes, expression of the receptor CD38 and of the kinase ZAP‐70 are independent markers of unfavorable outcome in CLL patients.Our group studies the role of CD38 in CLL not only as disease marker but also as pathogenetic agent. We showed that CD38 enhances proliferation of CLL cells through binding CD31, the ligand expressed by stromal and endothelial cells. Further, i) CD38+/ZAP‐70+ cells efficiently migrate towards CXCL12, ii) CD38 engagement leads to tyrosine phosphorylation of ZAP‐70 and iii) CD38 pathway is impaired in CD38+/ZAP‐70− CLL cells.Collectively, these data point to a role for the CD38/ZAP‐70 pathway in CLL migration towards CXCL12. Indeed, CXCL12‐mediated chemotaxis can be blocked by anti‐CD38 mAbs. Pre‐treatment of CLL with anti‐CD38 mAbs impairs CXCR4 signaling, as indicated by lack of Ca2+ mobilization, ERK1/2 activation and receptor internalization. Further, CD38 and CXCR4 are associated, as shown by confocal microscopy and co‐immunoprecipitation.In conclusion, these results offer biological evidence explaining why the combination of CD38 and ZAP‐70 results in a more efficient identification of CLL patients with aggressive disease. Further, they provide preliminary rationale for using anti‐CD38 ligands or inhibitors as adjunct therapeutic tools.
The X‐ray Structure of IAd in Complex with a Self Peptide Offers New Insights Into the Basis of Autoimmune Gastritis (AIG)
Authors: Revilleza Maria Jamela R , Levin Ditza , Mage Michael G , Teyton Luc , Robinson Howard , Shevach Ethan M , Natarajan Kannan , Margulies David H 
Published: June 2021

We developed a mouse model of autoimmune gastritis in which BALB/c mice expressing a transgenic αβ TCR, designated A51, spontaneously develop the disease. The TCR recognizes a 12‐mer peptide from the alpha subunit of gastric parietal cell H/K ATPase residues 889–900 (PLLCVGLRPQWE (H/K ATPase 889–900)), presented in the context of IAd. The disease, a model of human pernicious anemia, is characterized by slow and incomplete penetrance, anti‐gastric mucosal cell antibodies, inflammatory eosinophilic infiltrates, and parietal cell destruction. To investigate the contribution of MHC/peptide interactions to the initiation and maintenance of AIG, we generated a soluble recombinant version of the IAd/H/K ATPase889‐900 complex for biophysical and crystallographic analyses. We report here the 2.5 Å X‐ray structure of the complex. The structure was solved by molecular replacement and refined to Rfree of 28.9 by standard methods. The identification of peptide anchor residues and of solvent‐exposed side chains as T cell epitopic residues readily explains T cell proliferation data and off‐rate measurements of a panel of truncated, chimeric, and mutant peptides. This first structure of IAd in complex with an autoimmunogenic peptide reveals a heretofore unobserved peptide binding motif for IAd.This was supported by the Intramural Research Program of the NIAID/NIH.