1,721,355 research outputs found
Mac1 N40A protein is less stable in cells than are Mac1 WT or Mac1 N40D proteins.
No full textWestern blot analyses of cell lysates from A549 lung cancer cells lentivirally transduced with vectors expressing Strep-tagged Mac1 WT, Mac1 N40D or Mac1 N40A proteins. β-actin is shown as a loading control. The blot is representative of three independent replicates. (TIF)</p
Transfer of MAC1 to HT1080 cells.
No full text(a) MAC1 was introduced into HT1080 cells using PEG-MMCT (PEG) or Am-MMCT (Am). MAC1-transferred G418-resistant HT1080 clones were stained with crystal violet. Representative images of the plates are shown. (b) Comparison of total colony numbers generated by the PEG-MMCT and Am-MMCT methods. Each value represents the mean ± SD (n = 3; *P < 0.01). (c) FISH analysis of a MAC1-transferred HT1080 clone obtained by Am-MMCT. MAC1 (arrow) was detected with a mouse major satellite DNA probe (red). Human chromosomes were detected using a human Cot-1 probe (Green). The sample was counterstained with DAPI to visualize chromosomes (blue). Inset: high magnification image of MAC1. (d) Phase (left panel) or fluorescent (right panel) images of MAC1-transferred HT1080 clone. Scale bar, 100 μm. (e) Genomic PCR using primer sets specific for EGFP expression cassettes of lentivirus vector (EGFP (Lv)) or MAC1 (EGFP (MAC1)). Genomic DNA prepared from indicated cells was used as template. AmCHO-MAC1, AmCHO cells carrying MAC1; HT1080-MAC1, MAC1-transferred HT1080 cells obtained by Am-MMCT. (f) Genomic PCR using a primer set for 3´HPRT cassette of MAC1.</p
Transfer of MAC1 to NIH3T3 cells.
No full text(a) MAC1 was introduced into NIH3T3 cells using PEG-MMCT (PEG), Eco-MMCT (Eco), or Am-MMCT (Am). MAC1-transferred NIH3T3 cells were selected with G418, and colonies were stained with crystal violet. Representative images of the plates are shown. (b) Comparison of total colony numbers generated using the three MMCT methods. (c) FISH analysis of a MAC1-transferred NIH3T3 clone obtained by Eco-MMCT. Chromosomes of mouse origin were detected using a mouse major satellite probe (red). MAC1 (arrow) was identified with a Neo gene probe (green). The sample was counterstained with DAPI to visualize chromosomes (blue). Inset: high magnification image of MAC1. (d) Phase (upper panel) or fluorescent (lower panel) images of MAC1-transferred NIH3T3 clone. Scale bar, 100 μm. (e) Genomic PCR using primer sets specific for EGFP expression cassettes of lentivirus vector (EGFP (Lv)) or MAC1 (EGFP (MAC1)). Genomic DNA prepared from indicated cells was used as template. EcoCHO-MAC1, EcoCHO cells carrying MAC1; NIH3T3-MAC1, MAC1-transferred NIH3T3 cells obtained by Eco-MMCT. (f) Genomic PCR using a primer set for 3´HPRT cassette of MAC1. (g) Efficiency of retro-MMCT using cryopreserved microcells. MAC1 was introduced to NIH3T3 cells using cryopreserved microcells and all three MMCT methods. Each value represents the mean ± SD (n = 3; *P P < 0.01).</p
Talin and kindlin : batman and robin of Mac1 integrin activation?
No full textMacrophage 1 (Mac1) integrin is a leukocyte-restricted adhesion molecule highly expressed on phagocytes and is essential in the development of immune responses. In a normal physiological condition, activation of Mac1 is initiated via inside-out signaling. The final common step of inside-out signaling is the binding of a cytoplasmic protein talin to the integrin β cytoplasmic tail. Talin-binding unclasps the proximal region of αβ cytoplasmic tails, rendering integrin activation. Recently, another family of cytoplasmic protein kindlin is observed to bind another region along integrin β cytoplasmic tail and cooperate with talin as it boosted up the ligand affinity of platelet integrin and leukocyte function adhesion 1 (LFA-1) integrin upon activation. Thus, this study aims to clarify if kindlin-3 also serves as co-activator of Mac1 in the presence of talin. The findings in 293T transient transfection system suggested that kindlin-3 is likely to serve as the co-activator of Mac1 in the presence of talin because kindlin-3 long (K3L) enhanced the ligand-binding of Mac1 induced by talin head domain (talin-HD), but K3L alone did not activate Mac1. Any possible involvement of kindlin-3 in the cell spreading remains to be further clarified.Bachelor of Science in Biological Science
The C. neoformans Mac1 Protein Is Polymorphic
No full text<div><p>Translated amino acid sequences for Mac1 from strains B3501α and JEC21α (congenic to B3502<b>a</b>) are depicted. CNG02270 contains a copper-fist DNA binding domain at the N-terminus that is conserved between copper-regulating transcription factors, such as Mac1 of S. cerevisiae and Cuf1 of S. pombe. It also shares a copper-binding motif at the C-terminal region with S. pombe Cuf1 (328–342aa) that is important for activity.</p><p>^, sequences underlined with this mark are the predicted copper-Fist DNA binding domain.</p><p>#, cysteine and histidine residues conserved between Cuf1 of S. pombe and Mac1 of <i>C. neoformans.</i></p><p>Cn, <i>C. neoformans;</i> Sc, <i>S. cerevisiae;</i> Sp, S. pombe.</p></div
SARS-CoV-2 Mac1 is an essential virulence factor
Get PDFSeveral coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in this set of proteins is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and interferon-stimulated gene (ISG) expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target.National Institutes of Health (NIH) grant P20GM103648 (RC) National Institutes of Health (NIH) grant 2P01AI060699 (LE) National Institutes of Health (NIH) grant P20GM113117 (ARF) National Institutes of Health (NIH) grant K22AI134993 (ARF) National Institutes of Health (NIH) grant R35GM138029 (ARF) National Science Foundation (NSF) grant 2135167 (RLU) University of Kansas General Research Fund (GRF) and Start-up funds (ARF) NIH Graduate Training at the Biology-Chemistry Interface grant T32GM132061 (CMK) University of Kansas College of Liberal Arts and Sciences Graduate Research Fellowship (CMK) Government of Spain (PID2019-107001RB-I00 AEI/FEDER, UE) LE European Commission (H2020-SC1-2019, ISOLDA Project nº 848166-2) LEN
Mac1 Is a QTG Regulating Growth, Melanization, and Filamentation of C. neoformans
No full text<p>Wild-type strain XL304α and its <i>mac1</i> mutant were cultured on YPD medium with the indicated concentrations of BCS or copper sulfate at 22 °C (A) and 39 °C (B). The <i>mac1</i> mutant showed hypersensitivity under copper-limiting and copper-rich conditions at both temperatures. Wild-type and the <i>mac1</i> mutant were cultured on L-DOPA medium with the indicated concentrations of BCS or copper sulfate at 22 °C for induction of melanin production (C). The melanin production in <i>mac1</i> mutant is also hypersensitive to copper in the medium, showing a bell-shaped response similar to growth (C). Wild-type and the <i>mac1</i> mutant were cultured on V8 medium (pH 7.0) with the indicated concentrations of BCS or copper sulfate at 22 °C for induction of filamentation (D). Filamentation of the <i>mac1</i> mutant is highly sensitive to copper ions in the medium (D). <i>MAC1</i> allele exchange indicates different functions of the two alleles (E). Wild-type XL304α, wild-type JEC21α, JEC21α <i>mac1</i> mutant, and transgenic JEC21α <i>mac1</i> mutant with the <i>MAC1</i> XL304α allele were cultured on L-DOPA medium at 22 °C with the indicated concentrations of BCS or copper sulfate (E).</p
Putative Mac1-interacting genes identified by affinity purification.
No full text<p>Putative Mac1-interacting genes identified by affinity purification.</p
BiFC assays for the Cap1-Mac1 interaction.
No full text<p>Vegetative hyphae, conidia, and appressoria of transformant CMB14 expressing the <i>CAP1</i>-NYFP and <i>MAC1</i><sup>CT</sup>-CYFP constructs were examined by DIC and epifluorescence microscopy. Bar = 10 µm.</p
Microglial MAC1 receptor and PI3K are essential in mediating β-amyloid peptide-induced microglial activation and subsequent neurotoxicity
Get PDFAbstract Background β-Amyloid peptide (Aβ) is a major protein in the brain associated with Alzheimer's and Parkinson's diseases. The purpose of this study was to investigate the role of macrophage antigen-1 (MAC1) receptor, an integrin scavenger receptor in microglia, and subsequent signaling events in mediating Aβ-induced neurotoxicity. We have previously reported that NADPH oxidase (PHOX) on microglia and superoxide produced by PHOX are critical for Aβ-induced loss of dopaminergic neurons. However, the upstream signaling pathway of superoxide production remains unclear. Methods For the in vitro study, mesencephalic neuron-glia cultures and microglia-enriched cultures from mice deficient in the MAC1 receptor (MAC1-/-) and wild type controls were used to investigate the role of MAC1 receptor in Aβ-induced neurotoxicity and the role of phosphoinositide-3 kinase (PI3K) in the signal pathway between MAC1 receptor and PHOX. For the in vivo study, Aβ was injected into the substantia nigra of MAC1-/- mice and wild type mice to confirm the role of MAC1 receptor. Results We found that Aβ-induced activation of microglia, activation of PHOX, generation of superoxide and other reactive oxygen species, and loss of dopaminergic neurons were decreased in MAC1-/- cultures compared to MAC1+/+ cultures. In MAC1-/- mice, dopaminergic neuron loss in response to Aβ injection into the substantia nigra was reduced relative to MAC1+/+ mice. Thus, MAC1 receptor-mediated PHOX activation and increased superoxide production are associated with Aβ-induced neurotoxicity. PI3K activation was one downstream step in MAC1 signaling to PHOX and played an important role in Aβ-induced neurotoxicity. In microglia-enriched cultures from MAC1-/- mice, Aβ-induced activation of PI3K (phosphorylation of target proteins and PIP3 production) was reduced relative to MAC1+/+ cultures. Conclusions Taken together, our data demonstrate that Aβ activates MAC1 receptor to increase the activity of PI3K, which in turn phosphorylates p47phox, triggers the translocation of cytosolic subunits of PHOX to microglia membrane, increases PHOX activation and the subsequent production of superoxide and causes neurotoxicity.</p
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