C-tag TNF: a reporter system to study TNF shedding [Methods and Resources]
TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow...
Source: Journal of Biological Chemistry - December 25, 2020 Category: Chemistry Authors: Francesca Pinci, Moritz M. Gaidt, Christophe Jung, Gunnar Kuut, Margaret A. Jackson, Stefan Bauernfried, Veit Hornung Tags: Immunology Source Type: research

MicroRNA-98 reduces nerve growth factor expression in nicotine-induced airway remodeling [Gene Regulation]
Evolving evidence suggests that nicotine may contribute to impaired asthma control by stimulating expression of nerve growth factor (NGF), a neurotrophin associated with airway remodeling and airway hyperresponsiveness. We explored the hypothesis that nicotine increases NGF by reducing lung fibroblast (LF) microRNA-98 (miR-98) and PPARγ levels, thus promoting airway remodeling. Levels of NGF, miR-98, PPARγ, fibronectin 1 (FN1), endothelin-1 (EDN1, herein referred to as ET-1), and collagen (COL1A1 and COL3A1) were measured in human LFs isolated from smoking donors, in mouse primary LFs exposed to nicotine (50 μg/ml), and...
Source: Journal of Biological Chemistry - December 25, 2020 Category: Chemistry Authors: Cherry Wongtrakool, Junsuk Ko, Andrew J. Jang, Kora Grooms, Sarah Chang, Cory Sylber, Beata Kosmider, Karim Bahmed, Michael R. Blackburn, Roy L. Sutliff, C. Michael Hart, Changwon Park, Toru Nyunoya, Michael J. Passineau, Qing Lu, Bum-Yong Kang Tags: Molecular Bases of Disease Source Type: research

Quantitative phosphoproteomic analysis reveals involvement of PD-1 in multiple T cell functions [Signal Transduction]
Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell–mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell–mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation ...
Source: Journal of Biological Chemistry - December 25, 2020 Category: Chemistry Authors: Anna S. Tocheva, Michael Peled, Marianne Strazza, Kieran R. Adam, Shalom Lerrer, Shruti Nayak, Inbar Azoulay-Alfaguter, Connor J. R. Foster, Elliot A. Philips, Benjamin G. Neel, Beatrix Ueberheide, Adam Mor Tags: Immunology Source Type: research

Murine GFP-Mx1 forms nuclear condensates and associates with cytoplasmic intermediate filaments: Novel antiviral activity against VSV [Immunology]
Type I and III interferons induce expression of the “myxovirus resistance proteins” MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles (“biomolecular condensates”). In the present study, we investigated whether nuclear murine Mx1 st...
Source: Journal of Biological Chemistry - December 25, 2020 Category: Chemistry Authors: Pravin B. Sehgal, Huijuan Yuan, Mia F. Scott, Yan Deng, Feng-Xia Liang, Andrzej Mackiewicz Tags: Cell Biology Source Type: research

High temperature promotes amyloid {beta}-protein production and {gamma}-secretase complex formation via Hsp90 [Neurobiology]
Alzheimer's disease (AD) is characterized by neuronal loss and accumulation of β-amyloid-protein (Aβ) in the brain parenchyma. Sleep impairment is associated with AD and affects about 25–40% of patients in the mild-to-moderate stages of the disease. Sleep deprivation leads to increased Aβ production; however, its mechanism remains largely unknown. We hypothesized that the increase in core body temperature induced by sleep deprivation may promote Aβ production. Here, we report temperature-dependent regulation of Aβ production. We found that an increase in temperature, from 37 °C to 39 °C, significantly increased A...
Source: Journal of Biological Chemistry - December 25, 2020 Category: Chemistry Authors: Arshad Ali Noorani, Hitoshi Yamashita, Yuan Gao, Sadequl Islam, Yang Sun, Tomohisa Nakamura, Hiroyuki Enomoto, Kun Zou, Makoto Michikawa Tags: Molecular Bases of Disease Source Type: research

Exofacial membrane composition and lipid metabolism regulates plasma membrane P4-ATPase substrate specificity [Lipids]
The plasma membrane of a cell is characterized by an asymmetric distribution of lipid species across the exofacial and cytofacial aspects of the bilayer. Regulation of membrane asymmetry is a fundamental characteristic of membrane biology and is crucial for signal transduction, vesicle transport, and cell division. The type IV family of P-ATPases, or P4-ATPases, establishes membrane asymmetry by selection and transfer of a subset of membrane lipids from the lumenal or exofacial leaflet to the cytofacial aspect of the bilayer. It is unclear how P4-ATPases sort through the spectrum of membrane lipids to identify their desire...
Source: Journal of Biological Chemistry - December 25, 2020 Category: Chemistry Authors: Bhawik Kumar Jain, Bartholomew P. Roland, Todd R. Graham Tags: Membrane Biology Source Type: research

Coronavirus infection and PARP expression dysregulate the NAD metabolome: An actionable component of innate immunity [Molecular Bases of Disease]
Poly(ADP-ribose) polymerase (PARP) superfamily members covalently link either a single ADP-ribose (ADPR) or a chain of ADPR units to proteins using NAD as the source of ADPR. Although the well-known poly(ADP-ribosylating) (PARylating) PARPs primarily function in the DNA damage response, many noncanonical mono(ADP-ribosylating) (MARylating) PARPs are associated with cellular antiviral responses. We recently demonstrated robust up-regulation of several PARPs following infection with murine hepatitis virus (MHV), a model coronavirus. Here we show that SARS-CoV-2 infection strikingly up-regulates MARylating PARPs and induces t...
Source: Journal of Biological Chemistry - December 25, 2020 Category: Chemistry Authors: Collin D. Heer, Daniel J. Sanderson, Lynden S. Voth, Yousef M. O. Alhammad, Mark S. Schmidt, Samuel A. J. Trammell, Stanley Perlman, Michael S. Cohen, Anthony R. Fehr, Charles Brenner Tags: Metabolism Source Type: research

Inhibition of the SUV4-20 H1 histone methyltransferase increases frataxin expression in Friedreich's ataxia patient cells [Gene Regulation]
The molecular mechanisms of reduced frataxin (FXN) expression in Friedreich's ataxia (FRDA) are linked to epigenetic modification of the FXN locus caused by the disease-associated GAA expansion. Here, we identify that SUV4-20 histone methyltransferases, specifically SUV4-20 H1, play an important role in the regulation of FXN expression and represent a novel therapeutic target. Using a human FXN–GAA–Luciferase repeat expansion genomic DNA reporter model of FRDA, we screened the Structural Genomics Consortium epigenetic probe collection. We found that pharmacological inhibition of the SUV4-20 methyltransferases by the to...
Source: Journal of Biological Chemistry - December 25, 2020 Category: Chemistry Authors: Gabriela Vilema–Enriquez, Robert Quinlan, Peter Kilfeather, Roberta Mazzone, Saba Saqlain, Irene del Molino del Barrio, Annalidia Donato, Gabriele Corda, Fengling Li, Masoud Vedadi, Andrea H. Nemeth, Paul E. Brennan, Richard Wade–Martins Tags: Molecular Bases of Disease Source Type: research

HIV-1 Gag release from yeast reveals ESCRT interaction with the Gag N-terminal protein region [Molecular Bases of Disease]
The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but...
Source: Journal of Biological Chemistry - December 25, 2020 Category: Chemistry Authors: Birgit Meusser, Bettina Purfuerst, Friedrich C. Luft Tags: Cell Biology Source Type: research

Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology [Computational Biology]
In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibiti...
Source: Journal of Biological Chemistry - December 25, 2020 Category: Chemistry Authors: Marcus J. G. W. Ladds, Gergana Popova, Andres Pastor–Fernandez, Srinivasaraghavan Kannan, Ingeborg M. M. van Leeuwen, Maria Hakansson, Bȷorn Walse, Fredrik Tholander, Ravi Bhatia, Chandra S. Verma, David P. Lane, Sonia Lain Tags: Cell Biology Source Type: research

Identification of compounds that bind the centriolar protein SAS-6 and inhibit its oligomerization [Computational Biology]
Centrioles are key eukaryotic organelles that are responsible for the formation of cilia and flagella, and for organizing the microtubule network and the mitotic spindle in animals. Centriole assembly requires oligomerization of the essential protein spindle assembly abnormal 6 (SAS-6), which forms a structural scaffold templating the organization of further organelle components. A dimerization interaction between SAS-6 N-terminal “head” domains was previously shown to be essential for protein oligomerization in vitro and for function in centriole assembly. Here, we developed a pharmacophore model allowing us to assemb...
Source: Journal of Biological Chemistry - December 25, 2020 Category: Chemistry Authors: Julia M. C. Busch, Minos-Timotheos Matsoukas, Maria Musgaard, Georgios A. Spyroulias, Philip C. Biggin, Ioannis Vakonakis Tags: Molecular Biophysics Source Type: research

Molecular characterization of the RNA-protein complex directing -2/-1 programmed ribosomal frameshifting during arterivirus replicase expression [Protein Structure and Folding]
Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs −1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical −1 and −2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich mo...
Source: Journal of Biological Chemistry - December 25, 2020 Category: Chemistry Authors: Ankoor Patel, Emmely E. Treffers, Markus Meier, Trushar R. Patel, Jorg Stetefeld, Eric J. Sniȷder, Brian L. Mark Tags: Editors ' Picks Source Type: research

A kinetic dissection of the fast and superprocessive kinesin-3 KIF1A reveals a predominant one-head-bound state during its chemomechanical cycle [Molecular Biophysics]
The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to WT KIF1A. We established that the KIF1A forward step ...
Source: Journal of Biological Chemistry - December 25, 2020 Category: Chemistry Authors: Taylor M. Zaniewski, Allison M. Gicking, John Fricks, William O. Hancock Tags: Editors ' Picks Source Type: research

Withdrawal: Exercise increases mitochondrial PGC-1{alpha} content and promotes nuclear-mitochondrial cross-talk to coordinate mitochondrial biogenesis. [Withdrawals/Retractions]
This article has been withdrawn by the authors. In Fig. 6B the top two panels, corresponding to TOMM22 and Bcl-2, with trypsin treatment samples presented background issues. In Fig. 5, the H2B and VDAC immunoblot panels were duplicated and flipped vertically. The immunoblots for VDAC in Fig. 7A and the VDAC panels in Fig. 8B were duplicated and represented different experimental conditions. The immunoblots for Tfam (IP: PGC-1a) in Fig. 7A and the nuclear PGC-1a in Fig. 5 were flipped vertically. The immunoblots for PGC-1a (IP: Tfam) in Fig. 7B and PGC-1a (Input: D-Loop) in Fig. 8 were flipped horizontally and duplicated. D...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Adeel Safdar, Jonathan P. Little, Andrew J. Stokl, Bart P. Hettinga, Mahmood Akhtar, Mark A. Tarnopolsky Tags: Withdrawals/Retractions Source Type: research

Withdrawal: ETS-1 protein regulates vascular endothelial growth factor-induced matrix metalloproteinase-9 and matrix metalloproteinase-13 expression in human ovarian carcinoma cell line SKOV-3. [Withdrawals/Retractions]
VOLUME 287 (2012) PAGES 15001–15015The article has been withdrawn by the authors. The authors were made aware that the panel corresponding to lanes 3 and 4 of the total Akt immunoblot in Fig. 3A was reused as a GAPDH panel in Fig. 6B. In addition, the panel corresponding to the GAPDH immunoblot in Fig. 3A was inappropriately manipulated to reduce background noise. The authors responded that all the experiments were executed appropriately, and the data obtained at that time or by subsequent experiments support the original conclusion of the paper. Furthermore, the authors state that these issues do not affect the accuracy...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Sonali Ghosh, Moitri Basu, Sib Sankar Roy Tags: Withdrawals/Retractions Source Type: research