scholarly journals Molecular mechanism of dynein recruitment to kinetochores by the Rod–Zw10–Zwilch complex and Spindly

2017 ◽  
Vol 216 (4) ◽  
pp. 943-960 ◽  
Author(s):  
José B. Gama ◽  
Cláudia Pereira ◽  
Patrícia A. Simões ◽  
Ricardo Celestino ◽  
Rita M. Reis ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Intracellular Transport ◽  
Molecular Motor ◽  
Coiled Coil ◽  
Common Mechanism ◽  
Mitotic Chromosomes ◽  
Kinetochore Region ◽  
Pointed End

The molecular motor dynein concentrates at the kinetochore region of mitotic chromosomes in animals to accelerate spindle microtubule capture and to control spindle checkpoint signaling. In this study, we describe the molecular mechanism used by the Rod–Zw10–Zwilch complex and the adaptor Spindly to recruit dynein to kinetochores in Caenorhabditis elegans embryos and human cells. We show that Rod’s N-terminal β-propeller and the associated Zwilch subunit bind Spindly’s C-terminal domain, and we identify a specific Zwilch mutant that abrogates Spindly and dynein recruitment in vivo and Spindly binding to a Rod β-propeller–Zwilch complex in vitro. Spindly’s N-terminal coiled-coil uses distinct motifs to bind dynein light intermediate chain and the pointed-end complex of dynactin. Mutations in these motifs inhibit assembly of a dynein–dynactin–Spindly complex, and a null mutant of the dynactin pointed-end subunit p27 prevents kinetochore recruitment of dynein–dynactin without affecting other mitotic functions of the motor. Conservation of Spindly-like motifs in adaptors involved in intracellular transport suggests a common mechanism for linking dynein to cargo.

scholarly journals Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan

Microbiology ◽  
2014 ◽  
Vol 160 (10) ◽  
pp. 2157-2169 ◽  
Author(s):  
Sudarson Sundarrajan ◽  
Junjappa Raghupatil ◽  
Aradhana Vipra ◽  
Nagalakshmi Narasimhaswamy ◽  
Sanjeev Saravanan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Mechanism Of Action ◽  
Catalytic Domain ◽  
Antibiotic Sensitivity ◽  
High Sensitivity ◽  
Common Mechanism ◽  
Cross Bridge ◽  
Sensitivity Pattern

P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other β-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Min Zheng ◽  
David R. Knowles ◽  
Ronald K.H. Liem
Keyword(s):  
Calcium Binding ◽  
Coiled Coil ◽  
Specific Protein ◽  
Binding Domain ◽  
Full Length ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

scholarly journals PGC7 suppresses TET3 for protecting DNA methylation

2013 ◽  
Vol 42 (5) ◽  
pp. 2893-2905 ◽  
Author(s):  
Chunjing Bian ◽  
Xiaochun Yu
Keyword(s):  
Dna Methylation ◽  
Molecular Mechanism ◽  
Biological Process ◽  
Cpg Islands ◽  
Binding Motifs ◽  
Genome Wide Analysis ◽  
Dna Motif ◽  
Genome Wide

Abstract Ten-eleven translocation (TET) family enzymes convert 5-methylcytosine to 5-hydroxylmethylcytosine. However, the molecular mechanism that regulates this biological process is not clear. Here, we show the evidence that PGC7 (also known as Dppa3 or Stella) interacts with TET2 and TET3 both in vitro and in vivo to suppress the enzymatic activity of TET2 and TET3. Moreover, lacking PGC7 induces the loss of DNA methylation at imprinting loci. Genome-wide analysis of PGC7 reveals a consensus DNA motif that is recognized by PGC7. The CpG islands surrounding the PGC7-binding motifs are hypermethylated. Taken together, our study demonstrates a molecular mechanism by which PGC7 protects DNA methylation from TET family enzyme-dependent oxidation.

Abstract MP03: ROCK2 Specific Inhibition Attenuates Angiotensin II-induced Hypertension In Mice

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Daniel J Fehrenbach ◽  
Meena S Madhur
Keyword(s):  
Blood Pressure ◽  
T Cell ◽  
Angiotensin Ii ◽  
Repeated Measures ◽  
Flow Cytometric Analysis ◽  
Coiled Coil ◽  
Ang Ii ◽  
Induced Hypertension

Hypertension, or an elevated blood pressure, is the primary modifiable risk factor for cardiovascular disease, the number one cause of mortality worldwide. We previously demonstrated that Th17 activation and interleukin 17A (IL-17A)/IL-21 production is integral for the full development of a hypertensive phenotype as well as the renal and vascular damage associated with hypertension. Rho-associated coiled-coil containing protein Kinase 2 (ROCK2) serves as a molecular switch upregulating Th17 and inhibiting regulatory T cell (Treg) differentiation. We hypothesize that hypertension is characterized by excessive T cell ROCK2 activation leading to increased Th17/Treg ratios and ultimately end-organ damage. We first showed in vitro that KD025, an experimental orally bioavailable ROCK2 inhibitor inhibits Th17 cell proliferation and IL-17A/IL-21 production. To determine if hypertensive stimuli such as endothelial stretch increases T cell ROCK2 expression, we cultured human aortic endothelial cells exposed to 5% (normotensive) or 10% (hypertensive) stretch with circulating human T cells and HLA-DR+ antigen presenting cells. Hypertensive stretch increased T cell ROCK2 expression 2-fold. We then tested the effect of ROCK2 inhibition with KD025 (50mg/kg i.p. daily) in vivo on angiotensin II (Ang II)-induced hypertension. Treatment with KD025 significantly attenuated the hypertensive response within 1 week of Ang II treatment (systolic blood pressure: 139± 8 vs 108±7mmHg) and this persisted for the duration of the 4 week study reaching blood pressures 20 mmHg lower (135±13mmHg) than vehicle treated mice (158±4mmHg p<0.05 effect of treatment 2-way Repeated Measures ANOVA). Flow cytometric analysis of tissue infiltrating leukocytes revealed that KD025 treatment increased Treg/Th17 ratios in the kidney (0.61±0.03 vs 0.79±0.08, p<0.05 student’s t-test). Thus, T cell ROCK2 may be a novel therapeutic target for the treatment of hypertension.

Abstract 12231: PECAM1 is Essential for Flow-mediated Gab1 Tyrosine Phosphorylation and Signaling in vitro and in vivo

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Suowen Xu ◽  
Marina Koroleva ◽  
Keigi Fujiwara ◽  
Zheng Gen Jin
Keyword(s):  
Tyrosine Phosphorylation ◽  
Wheel Running ◽  
Tyrosine Phosphatase ◽  
Common Mechanism ◽  
Membrane Localization ◽  
No Production ◽  
Voluntary Wheel Running ◽  
Voluntary Wheel

Introduction: Impaired activation of endothelial nitric oxide (NO) synthase (eNOS) and ensued NO production is a common mechanism of various cardiovascular pathologies, including hypertension and atherosclerosis. Specific signaling cascades, generated by vascular endothelial cells (ECs) in response to laminar flow, modulate EC structure and functions, NO production in particular. We have previously shown that flow-stimulated Gab1 (Grb2-associated binder-1) tyrosine phosphorylation mediates eNOS activation. However, the upstream mechanism that regulates Gab1 tyrosine phosphorylation remains unclear. Hypothesis: We hypothesized that platelet endothelial cell adhesion molecule-1 (PECAM1), a key molecule in an endothelial mechanosensing complex, specifically mediates Gab1 tyrosine phosphorylation and its downstream Akt and eNOS activation in ECs upon flow rather than hepatocyte growth factor (HGF) stimulation. Methods: Western blot, en face staining and voluntary wheel running. Results: Small interfering RNA (siRNA) targeting PECAM1 abolished flow- but not HGF-induced Gab1 tyrosine phosphorylation and Akt, eNOS activation as well as Gab1 membrane translocation. Protein-tyrosine phosphatase SHP2, which has been shown to interact with Gab1, was involved in a flow signaling pathway as well as HGF-induced signaling, as SHP2 siRNA diminished the flow- and HGF-induced Gab1 tyrosine phosphorylation, membrane localization and downstream signaling. Pharmacological inhibition of PI3K by LY294002 decreased flow, but not HGF-mediated Gab1 phosphorylation and membrane localization as well as eNOS activation. Finally, we observed that flow-mediated Gab1 and eNOS phosphorylation in vivo induced by voluntary wheel running was reduced in PECAM1 knockout mice. Conclusions: These results demonstrate a specific role of PECAM1 in flow-mediated Gab1 tyrosine phosphorylation and eNOS signaling in ECs

NUANCE, a giant protein connecting the nucleus and actin cytoskeleton

2002 ◽  
Vol 115 (15) ◽  
pp. 3207-3222 ◽  
Author(s):  
Yen-Yi Zhen ◽  
Thorsten Libotte ◽  
Martina Munck ◽  
Angelika A. Noegel ◽  
Elena Korenbaum
Keyword(s):  
Actin Cytoskeleton ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
Peripheral Blood Lymphocytes ◽  
Binding Domain ◽  
Actin Binding ◽  
Actin Binding Domain ◽  
Microfilament System

NUANCE (NUcleus and ActiN Connecting Element) was identified as a novel protein with an α-actinin-like actin-binding domain. A human 21.8 kb cDNA of NUANCE spreads over 373 kb on chromosome 14q22.1-q22.3. The cDNA sequence predicts a 796 kDa protein with an N-terminal actin-binding domain, a central coiled-coil rod domain and a predicted C-terminal transmembrane domain. High levels of NUANCE mRNA were detected in the kidney, liver,stomach, placenta, spleen, lymphatic nodes and peripheral blood lymphocytes. At the subcellular level NUANCE is present predominantly at the outer nuclear membrane and in the nucleoplasm. Domain analysis shows that the actin-binding domain binds to Factin in vitro and colocalizes with the actin cytoskeleton in vivo as a GFP-fusion protein. The C-terminal transmembrane domain is responsible for the targeting the nuclear envelope. Thus, NUANCE is the firstα-actinin-related protein that has the potential to link the microfilament system with the nucleus.

scholarly journals Unraveling the Novel Protective Effect of Patchouli Alcohol Against Helicobacter pylori-Induced Gastritis: Insights Into the Molecular Mechanism in vitro and in vivo

2018 ◽  
Vol 9 ◽  
Author(s):  
Da-wei Lian ◽  
Yi-fei Xu ◽  
Wen-kang Ren ◽  
Li-jun Fu ◽  
Fang-jun Chen ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Protective Effect ◽  
Molecular Mechanism ◽  
The Novel ◽  
Patchouli Alcohol

scholarly journals Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

2002 ◽  
Vol 159 (6) ◽  
pp. 993-1004 ◽  
Author(s):  
Christine L. Humphries ◽  
Heath I. Balcer ◽  
Jessica L. D'Agostino ◽  
Barbara Winsor ◽  
David G. Drubin ◽  
...  
Keyword(s):  
Binding Protein ◽  
Coiled Coil ◽  
Actin Binding ◽  
Complex Activity ◽  
Actin Binding Protein ◽  
Coiled Coil Domain ◽  
Allele Specific ◽  
And Function

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

scholarly journals Triple-Helical DNA in Drosophila Heterochromatin

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 227 ◽  
Author(s):  
Eduardo Gorab
Keyword(s):  
Nucleic Acids ◽  
Dna Sequences ◽  
Detection Methods ◽  
Triplex Dna ◽  
Thiazole Orange ◽  
Chromosomal Dna ◽  
Mitotic Chromosomes ◽  
Satellite Repeats

Polynucleotide chains obeying Watson-Crick pairing are apt to form non-canonical complexes such as triple-helical nucleic acids. From early characterization in vitro, their occurrence in vivo has been strengthened by increasing evidence, although most remain circumstantial particularly for triplex DNA. Here, different approaches were employed to specify triple-stranded DNA sequences in the Drosophila melanogaster chromosomes. Antibodies to triplex nucleic acids, previously characterized, bind to centromeric regions of mitotic chromosomes and also to the polytene section 59E of mutant strains carrying the brown dominant allele, indicating that AAGAG tandem satellite repeats are triplex-forming sequences. The satellite probe hybridized to AAGAG-containing regions omitting chromosomal DNA denaturation, as expected, for the intra-molecular triplex DNA formation model in which single-stranded DNA coexists with triplexes. In addition, Thiazole Orange, previously described as capable of reproducing results obtained by antibodies to triple-helical DNA, binds to AAGAG repeats in situ thus validating both detection methods. Unusual phenotype and nuclear structure exhibited by Drosophila correlate with the non-canonical conformation of tandem satellite arrays. From the approaches that lead to the identification of triple-helical DNA in chromosomes, facilities particularly provided by Thiazole Orange use may broaden the investigation on the occurrence of triplex DNA in eukaryotic genomes.

scholarly journals “mir152 hypomethylation, potentially triggered by embryonic hypoxia, as a common mechanism for non-syndromic cleft lip/palate”

2019 ◽  
Author(s):  
Lucas Alvizi ◽  
Luciano Abreu Brito ◽  
Bárbara Bischain ◽  
Camila Bassi Fernandes da Silva ◽  
Sofia Ligia Guimaraes Ramos ◽  
...  
Keyword(s):  
Cleft Lip ◽  
Differentially Methylated Region ◽  
Common Mechanism ◽  
Craniofacial Malformations ◽  
Predisposing Factor ◽  
Complex Disorder ◽  
Cleft Lip Palate ◽  
Functional Investigation

AbstractNon-syndromic cleft lip/palate (NSCLP), the most common human craniofacial malformations, is a complex disorder given its genetic heterogeneity and multifactorial component revealed by genetic, epidemiological and epigenetic findings. Association of epigenetic variations with NSCLP has been made, however still of little functional investigation. Here we combined a reanalysis of NSCLP methylome data with genetic analysis and used both in vitro and in vivo approaches to dissect the functional effects of epigenetic changes. We found a frequent differentially methylated region in mir152, hypomethylated in NSCLP cohorts (21-26%), leading to mir152 overexpression. In vivo analysis using zebrafish embryos revealed that mir152 upregulation leads to craniofacial impairment analogue to palatal defects. Also, we demonstrated that zebrafish embryonic hypoxia leads to mir152 upregulation combined with mir152 hypomethylation and also analogue palatal alterations. We therefore suggest mir152 hypomethylation, potentially induced by hypoxia in early development, as a novel and frequent predisposing factor to NSCLP.

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