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IRDye® 800CW Goat anti-Rabbit IgG (H + L) antibody

RRID:AB_2651127

The Rheumatoid Arthritis-Associated Citrullinome.

  • Tilvawala R
  • Cell Chem Biol
  • 2018 Jun 21

Literature context:


Abstract:

Increased protein citrullination is linked to various diseases including rheumatoid arthritis (RA), lupus, and cancer. Citrullinated autoantigens, a hallmark of RA, are recognized by anti-citrullinated protein antibodies (ACPAs) which are used to diagnose RA. ACPA-recognizing citrullinated enolase, vimentin, keratin, and filaggrin are also pathogenic. Here, we used a chemoproteomic approach to define the RA-associated citrullinome. The identified proteins include numerous serine protease inhibitors (Serpins), proteases and metabolic enzymes. We demonstrate that citrullination of antiplasmin, antithrombin, t-PAI, and C1 inhibitor (P1-Arg-containing Serpins) abolishes their ability to inhibit their cognate proteases. Citrullination of nicotinamide N-methyl transferase (NNMT) also abolished its methyltransferase activity. Overall, these data advance our understanding of the roles of citrullination in RA and suggest that extracellular protein arginine deiminase (PAD) activity can modulate protease activity with consequent effects on Serpin-regulated pathways. Moreover, our data suggest that inhibition of extracellular PAD activity will be therapeutically relevant.

Funding information:
  • NCI NIH HHS - K07 CA102327(United States)
  • NIGMS NIH HHS - R01 GM110394()
  • NIGMS NIH HHS - R35 GM118112()

[18F]FMPEP-d2 PET imaging shows age- and genotype-dependent impairments in the availability of cannabinoid receptor 1 in a mouse model of Alzheimer's disease.

  • Takkinen JS
  • Neurobiol. Aging
  • 2018 May 18

Literature context:


Abstract:

Contradictory findings on the role of the type 1 cannabinoid receptor (CB1R) during the pathogenesis of Alzheimer's disease (AD) have been reported. Here, we evaluated the CB1R brain profile in an AD mouse model using longitudinal positron emission tomography with an inverse agonist for CB1R, [18F]FMPEP-d2. APP/PS1-21 and wild-type (n = 8 in each group) mice were repeatedly imaged between 6 to 15 months of age, accompanied by brain autoradiography, western blot, and CB1R immunohistochemistry with additional mice. [18F]FMPEP-d2 positron emission tomography demonstrated lower (p < 0.05) binding ratios in the parietotemporal cortex and hippocampus of APP/PS1-21 mice compared with age-matched wild-type mice. Western blot demonstrated no differences between APP/PS1-21 and wild-type mice in the CB1R abundance, whereas significantly lower (p < 0.05) receptor expression was observed in male than female mice. The results provide the first demonstration that [18F]FMPEP-d2 is a promising imaging tool for AD research in terms of CB1R availability, but not expression. This finding may further facilitate the development of novel therapeutic approaches based on endocannabinoid regulation.

Funding information:
  • NIGMS NIH HHS - R01 GM077668-03S1(United States)

ZFP36 RNA-binding proteins restrain T cell activation and anti-viral immunity.

  • Moore MJ
  • Elife
  • 2018 May 31

Literature context:


Abstract:

Dynamic post-transcriptional control of RNA expression by RNA-binding proteins (RBPs) is critical during immune response. ZFP36 RBPs are prominent inflammatory regulators linked to autoimmunity and cancer, but functions in adaptive immunity are less clear. We used HITS-CLIP to define ZFP36 targets in mouse T cells, revealing unanticipated actions in regulating T-cell activation, proliferation, and effector functions. Transcriptome and ribosome profiling showed that ZFP36 represses mRNA target abundance and translation, notably through novel AU-rich sites in coding sequence. Functional studies revealed that ZFP36 regulates early T-cell activation kinetics cell autonomously, by attenuating activation marker expression, limiting T cell expansion, and promoting apoptosis. Strikingly, loss of ZFP36 in vivo accelerated T cell responses to acute viral infection and enhanced anti-viral immunity. These findings uncover a critical role for ZFP36 RBPs in restraining T cell expansion and effector functions, and suggest ZFP36 inhibition as a strategy to enhance immune-based therapies.

Funding information:
  • National Institutes of Health - NS034389()
  • National Institutes of Health - NS081706()
  • National Institutes of Health - R35NS097404()
  • NIGMS NIH HHS - GM27681(United States)

Inhibition of Dpp8/9 Activates the Nlrp1b Inflammasome.

  • Okondo MC
  • Cell Chem Biol
  • 2018 Mar 15

Literature context:


Abstract:

Val-boroPro (PT-100, Talabostat) induces powerful anti-tumor immune responses in syngeneic cancer models, but its mechanism of action has not yet been established. Val-boroPro is a non-selective inhibitor of post-proline-cleaving serine proteases, and the inhibition of the highly related cytosolic serine proteases Dpp8 and Dpp9 (Dpp8/9) by Val-boroPro was recently demonstrated to trigger an immunostimulatory form of programmed cell death known as pyroptosis selectively in monocytes and macrophages. Here we show that Dpp8/9 inhibition activates the inflammasome sensor protein Nlrp1b, which in turn activates pro-caspase-1 to mediate pyroptosis. This work reveals a previously unrecognized mechanism for activating an innate immune pattern recognition receptor and suggests that Dpp8/9 serve as an intracellular checkpoint to restrain Nlrp1b and the innate immune system.

Funding information:
  • NIAID NIH HHS - F32 AI07718501(United States)

Inhibition of Methyltransferase Setd7 Allows the In Vitro Expansion of Myogenic Stem Cells with Improved Therapeutic Potential.

  • Judson RN
  • Cell Stem Cell
  • 2018 Feb 1

Literature context:


Abstract:

The development of cell therapy for repairing damaged or diseased skeletal muscle has been hindered by the inability to significantly expand immature, transplantable myogenic stem cells (MuSCs) in culture. To overcome this limitation, a deeper understanding of the mechanisms regulating the transition between activated, proliferating MuSCs and differentiation-primed, poorly engrafting progenitors is needed. Here, we show that methyltransferase Setd7 facilitates such transition by regulating the nuclear accumulation of β-catenin in proliferating MuSCs. Genetic or pharmacological inhibition of Setd7 promotes in vitro expansion of MuSCs and increases the yield of primary myogenic cell cultures. Upon transplantation, both mouse and human MuSCs expanded with a Setd7 small-molecule inhibitor are better able to repopulate the satellite cell niche, and treated mouse MuSCs show enhanced therapeutic potential in preclinical models of muscular dystrophy. Thus, Setd7 inhibition may help bypass a key obstacle in the translation of cell therapy for muscle disease.

Funding information:
  • BLRD VA - I01 BX002324()
  • NCI NIH HHS - R01 CA073808(United States)
  • NIA NIH HHS - P01 AG036695()
  • NIAMS NIH HHS - R21 AR071039()
  • RRD VA - I01 RX001222()

Conserved Lipid and Small-Molecule Modulation of COQ8 Reveals Regulation of the Ancient Kinase-like UbiB Family.

  • Reidenbach AG
  • Cell Chem Biol
  • 2018 Feb 15

Literature context:


Abstract:

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small molecules for probing its activity in vivo. Here, we demonstrate that COQ8 possesses evolutionarily conserved ATPase activity that is activated by binding to membranes containing cardiolipin and by phenolic compounds that resemble CoQ pathway intermediates. We further create an analog-sensitive version of Coq8p and reveal that acute chemical inhibition of its endogenous activity in yeast is sufficient to cause respiratory deficiency concomitant with CoQ depletion. Collectively, this work defines lipid and small-molecule modulators of an ancient family of atypical kinase-like proteins and establishes a chemical genetic system for further exploring the mechanistic role of COQ8 in CoQ biosynthesis.

Funding information:
  • NHLBI NIH HHS - T32 HL007899()
  • NIAMS NIH HHS - 1R03AR062832(United States)
  • NIGMS NIH HHS - R01 GM112057()
  • NIGMS NIH HHS - R35 GM118110()
  • NIGMS NIH HHS - T32 GM008505()
  • NIGMS NIH HHS - T32 GM008692()

Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division.

  • Lee ME
  • Curr. Biol.
  • 2018 Jan 8

Literature context:


Abstract:

Connections between the protein kinases that function within complex cell polarity networks are poorly understood. Rod-shaped fission yeast cells grow in a highly polarized manner, and genetic screens have identified many protein kinases, including the CaMKK-like Ssp1 and the MARK/PAR-1 family kinase Kin1, that are required for polarized growth and cell shape, but their functional mechanisms and connections have been unknown [1-5]. We found that Ssp1 promotes cell polarity by phosphorylating the activation loop of Kin1. Kin1 regulates cell polarity and cytokinesis through unknown mechanisms [4-7]. We performed a large-scale phosphoproteomic screen and found that Kin1 phosphorylates itself and Pal1 to promote growth at cell tips, and these proteins are interdependent for localization to growing cell tips. Additional Kin1 substrates for cell polarity and cytokinesis (Tea4, Mod5, Cdc15, and Cyk3) were also phosphorylated by a second kinase, the DYRK family member Pom1 [8]. Kin1 and Pom1 were enriched at opposite ends of growing cells, and they phosphorylated largely non-overlapping sites on shared substrates. Combined inhibition of both Kin1and Pom1 led to synthetic defects in their shared substrates Cdc15 and Cyk3, confirming a non-redundant functional connection through shared substrates. These findings uncover a new Ssp1-Kin1 signaling pathway, and define its functional and mechanistic connection with Pom1 signaling for cell polarity and cytokinesis. These kinases are conserved in many eukaryotes including humans, suggesting that similar connections and mechanisms might operate in a broad range of cells.

Funding information:
  • NIAID NIH HHS - U01 AI074509(United States)
  • NIGMS NIH HHS - R01 GM099774()
  • NIGMS NIH HHS - R35 GM119455()
  • NIGMS NIH HHS - T32 GM008704()

Notch Signaling Regulates Differentiation and Steroidogenesis in Female Mouse Ovarian Granulosa Cells.

  • Prasasya RD
  • Endocrinology
  • 2018 Jan 1

Literature context:


Abstract:

The Notch pathway is a highly conserved juxtacrine signaling mechanism that is important for many cellular processes during development, including differentiation and proliferation. Although Notch is important during ovarian follicle formation and early development, its functions during the gonadotropin-dependent stages of follicle development are largely unexplored. We observed positive regulation of Notch activity and expression of Notch ligands and receptors following activation of the luteinizing hormone-receptor in prepubertal mouse ovary. JAG1, the most abundantly expressed Notch ligand in mouse ovary, revealed a striking shift in localization from oocytes to somatic cells following hormone stimulation. Using primary cultures of granulosa cells, we investigated the functions of Jag1 using small interfering RNA knockdown. The loss of JAG1 led to suppression of granulosa cell differentiation as marked by reduced expression of enzymes and factors involved in steroid biosynthesis, and in steroid secretion. Jag1 knockdown also resulted in enhanced cell proliferation. These phenotypes were replicated, although less robustly, following knockdown of the obligate canonical Notch transcription factor RBPJ. Intracellular signaling analysis revealed increased activation of the mitogenic phosphatidylinositol 3-kinase/protein kinase B and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways following Notch knockdown, with a mitogen-activated protein kinase kinase inhibitor blocking the enhanced proliferation observed in Jag1 knockdown granulosa cells. Activation of YB-1, a known regulator of granulosa cell differentiation genes, was suppressed by Jag1 knockdown. Overall, this study reveals a role of Notch signaling in promoting the differentiation of preovulatory granulosa cells, adding to the diverse functions of Notch in the mammalian ovary.

Funding information:
  • NIA NIH HHS - R56 AG016379(United States)
  • NICHD NIH HHS - P01 HD021921(United States)
  • NICHD NIH HHS - P50 HD028934(United States)
  • NIGMS NIH HHS - T32 GM008061(United States)

Age-Dependent Dopaminergic Neurodegeneration and Impairment of the Autophagy-Lysosomal Pathway in LRRK-Deficient Mice.

  • Giaime E
  • Neuron
  • 2017 Nov 15

Literature context:


Abstract:

LRRK2 mutations are the most common genetic cause of Parkinson's disease, but LRRK2's normal physiological role in the brain is unclear. Here, we show that inactivation of LRRK2 and its functional homolog LRRK1 results in earlier mortality and age-dependent, selective neurodegeneration. Loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and of noradrenergic neurons in the locus coeruleus is accompanied with increases in apoptosis, whereas the cerebral cortex and cerebellum are unaffected. Furthermore, selective age-dependent neurodegeneration is only present in LRRK-/-, not LRRK1-/- or LRRK2-/- brains, and it is accompanied by increases in α-synuclein and impairment of the autophagy-lysosomal pathway. Quantitative electron microscopy (EM) analysis revealed age-dependent increases of autophagic vacuoles in the SNpc of LRRK-/- mice before the onset of DA neuron loss. These findings revealed an essential role of LRRK in the survival of DA neurons and in the regulation of the autophagy-lysosomal pathway in the aging brain.

Funding information:
  • NINDS NIH HHS - P50 NS094733()
  • NINDS NIH HHS - R01 NS071251()
  • NINDS NIH HHS - R37 NS071251()

Epithelial cell adhesion molecule fragments and signaling in primary human liver cells.

  • Gerlach JC
  • J. Cell. Physiol.
  • 2017 Nov 19

Literature context:


Abstract:

Epithelial Cell Adhesion Molecule (EpCAM), or CD326, is a trans-membrane glycoprotein expressed by multiple normal epithelia as well as carcinoma. Human hepatic stem cells and bile duct epithelium of the liver are EpCAM positive. In tumor cell lines, its intracellular domain can be released after cleavage of the extracellular domain. Within the cell nucleus, it induces cell proliferation, but cleavage depends on cell contact. Fragments of various lengths have been described in tumor cells. Despite its described important role in proliferation in tumor cells, there is not much known about the expression and role of EpCAM fragments in primary human liver cells. Here, we demonstrate that EpCAM protein fragments and function are considerable different between tumor cells, normal fetal and adult liver cells. Contrary to previously reported findings in tumor cells, gene knockdown or treatment with an inhibitor of the cleavage enzyme ADAM17 (TACE) rather increased cell numbers in primary human fetal liver-derived EpCAM-positive cells. EpCAM fragment sizes were not affected by treatment with inhibitor. Knockdown of EPCAM gene expression by siRNA in sorted cells did not significantly affect proliferation-associated genes or cell numbers. The intracellular domain could not be detected within cell nuclei of fetal and adult liver cells. In conclusion, signaling through the intracellular domain of EpCAM appears to be a mechanism that induces proliferation specifically in tumorigenic cells but not in normal primary EpCAM-positive liver cells.

Direct Activation of BAX by BTSA1 Overcomes Apoptosis Resistance in Acute Myeloid Leukemia.

  • Reyna DE
  • Cancer Cell
  • 2017 Oct 9

Literature context:


Abstract:

The BCL-2 family protein BAX is a central mediator of apoptosis. Overexpression of anti-apoptotic BCL-2 proteins contributes to tumor development and resistance to therapy by suppressing BAX and its activators. We report the discovery of BTSA1, a pharmacologically optimized BAX activator that binds with high affinity and specificity to the N-terminal activation site and induces conformational changes to BAX leading to BAX-mediated apoptosis. BTSA1-induced BAX activation effectively promotes apoptosis in leukemia cell lines and patient samples while sparing healthy cells. BAX expression levels and cytosolic conformation regulate sensitivity to BTSA1. BTSA1 potently suppressed human acute myeloid leukemia (AML) xenografts and increased host survival without toxicity. This study provides proof-of-concept for direct BAX activation as a treatment strategy in AML.

Argonaute CLIP Defines a Deregulated miR-122-Bound Transcriptome that Correlates with Patient Survival in Human Liver Cancer.

  • Luna JM
  • Mol. Cell
  • 2017 Aug 3

Literature context:


Abstract:

MicroRNA-122, an abundant and conserved liver-specific miRNA, regulates hepatic metabolism and functions as a tumor suppressor, yet systematic and direct biochemical elucidation of the miR-122 target network remains incomplete. To this end, we performed Argonaute crosslinking immunoprecipitation (Argonaute [Ago]-CLIP) sequencing in miR-122 knockout and control mouse livers, as well as in matched human hepatocellular carcinoma (HCC) and benign liver tissue to identify miRNA target sites transcriptome-wide in two species. We observed a majority of miR-122 binding on 3' UTRs and coding exons followed by extensive binding to other genic and non-genic sites. Motif analysis of miR-122-dependent binding revealed a G-bulged motif in addition to canonical motifs. A large number of miR-122 targets were found to be species specific. Upregulation of several common mouse and human targets, most notably BCL9, predicted survival in HCC patients. These results broadly define the molecular consequences of miR-122 downregulation in hepatocellular carcinoma.

IL-22 Upregulates Epithelial Claudin-2 to Drive Diarrhea and Enteric Pathogen Clearance.

  • Tsai PY
  • Cell Host Microbe
  • 2017 Jun 14

Literature context:


Abstract:

Diarrhea is a host response to enteric pathogens, but its impact on pathogenesis remains poorly defined. By infecting mice with the attaching and effacing bacteria Citrobacter rodentium, we defined the mechanisms and contributions of diarrhea and intestinal barrier loss to host defense. Increased permeability occurred within 2 days of infection and coincided with IL-22-dependent upregulation of the epithelial tight junction protein claudin-2. Permeability increases were limited to small molecules, as expected for the paracellular water and Na+ channel formed by claudin-2. Relative to wild-type, claudin-2-deficient mice experienced severe disease, including increased mucosal colonization by C. rodentium, prolonged pathogen shedding, exaggerated cytokine responses, and greater tissue injury. Conversely, transgenic claudin-2 overexpression reduced disease severity. Chemically induced osmotic diarrhea reduced colitis severity and C. rodentium burden in claudin-2-deficient, but not transgenic, mice, demonstrating that claudin-2-mediated protection is the result of enhanced water efflux. Thus, IL-22-induced claudin-2 upregulation drives diarrhea and pathogen clearance.

Funding information:
  • NCRR NIH HHS - S10 RR025643()
  • NICHD NIH HHS - T32 HD007009()
  • NIDDK NIH HHS - F30 DK103511()
  • NIDDK NIH HHS - F32 DK009180()
  • NIDDK NIH HHS - K01 DK092381()
  • NIDDK NIH HHS - R01 DK061931()
  • NIDDK NIH HHS - R01 DK068271()
  • NIDDK NIH HHS - R24 DK099803()

Pyroptosis and Apoptosis Pathways Engage in Bidirectional Crosstalk in Monocytes and Macrophages.

  • Taabazuing CY
  • Cell Chem Biol
  • 2017 Apr 20

Literature context:


Abstract:

Pyroptosis is a lytic form of programmed cell death mediated by the inflammatory caspase-1, -4, and -5. We recently discovered that small-molecule inhibitors of the serine peptidases DPP8 and DPP9 (DPP8/9) induce pro-caspase-1-dependent pyroptosis in monocytes and macrophages. Notably, DPP8/9 inhibitors, unlike microbial agents, absolutely require caspase-1 to induce cell death. Therefore, DPP8/9 inhibitors are useful probes to study caspase-1 in cells. Here, we show that, in the absence of the pyroptosis-mediating substrate gasdermin D (GSDMD), caspase-1 activates caspase-3 and -7 and induces apoptosis, demonstrating that GSDMD is the only caspase-1 substrate that induces pyroptosis. Conversely, we found that, during apoptosis, caspase-3/-7 specifically block pyroptosis by cleaving GSDMD at a distinct site from the inflammatory caspases that inactivates the protein. Overall, this work reveals bidirectional crosstalk between apoptosis and pyroptosis in monocytes and macrophages, further illuminating the complex interplay between cell death pathways in the innate immune system.

Funding information:
  • NCI NIH HHS - P30 CA008748()

Ligand and Target Discovery by Fragment-Based Screening in Human Cells.

  • Parker CG
  • Cell
  • 2017 Jan 26

Literature context:


Abstract:

Advances in the synthesis and screening of small-molecule libraries have accelerated the discovery of chemical probes for studying biological processes. Still, only a small fraction of the human proteome has chemical ligands. Here, we describe a platform that marries fragment-based ligand discovery with quantitative chemical proteomics to map thousands of reversible small molecule-protein interactions directly in human cells, many of which can be site-specifically determined. We show that fragment hits can be advanced to furnish selective ligands that affect the activity of proteins heretofore lacking chemical probes. We further combine fragment-based chemical proteomics with phenotypic screening to identify small molecules that promote adipocyte differentiation by engaging the poorly characterized membrane protein PGRMC2. Fragment-based screening in human cells thus provides an extensive proteome-wide map of protein ligandability and facilitates the coordinated discovery of bioactive small molecules and their molecular targets.

Funding information:
  • NCI NIH HHS - R01 CA132630()
  • NIDDK NIH HHS - R24 DK099810()
  • NIH HHS - S10 OD016357()

Ebola Virus Glycoprotein with Increased Infectivity Dominated the 2013-2016 Epidemic.

  • Diehl WE
  • Cell
  • 2016 Nov 3

Literature context:


Abstract:

The magnitude of the 2013-2016 Ebola virus disease (EVD) epidemic enabled an unprecedented number of viral mutations to occur over successive human-to-human transmission events, increasing the probability that adaptation to the human host occurred during the outbreak. We investigated one nonsynonymous mutation, Ebola virus (EBOV) glycoprotein (GP) mutant A82V, for its effect on viral infectivity. This mutation, located at the NPC1-binding site on EBOV GP, occurred early in the 2013-2016 outbreak and rose to high frequency. We found that GP-A82V had heightened ability to infect primate cells, including human dendritic cells. The increased infectivity was restricted to cells that have primate-specific NPC1 sequences at the EBOV interface, suggesting that this mutation was indeed an adaptation to the human host. GP-A82V was associated with increased mortality, consistent with the hypothesis that the heightened intrinsic infectivity of GP-A82V contributed to disease severity during the EVD epidemic.

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR.

  • Kaplan M
  • Cell
  • 2016 Nov 17

Literature context:


Abstract:

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase.