scholarly journals Oligomerisation of THAP9 Transposase: Role of DNA and Amino-Terminal Domains

2020 ◽  
Author(s):  
Hiral M. Sanghavi ◽  
Sharmistha MAJUMDAR
Keyword(s):  
Protein Interaction ◽  
Coiled Coil ◽  
P Element ◽  
Oligomeric State ◽  
Amino Terminal ◽  
Coiled Coil Region ◽  
Interaction Partners ◽  
Catalytically Active ◽  
Functional Relevance

Abstract BackgroundActive DNA transposases like the Drosophila P element transposase (DmTNP) undergo oligomerisation as a prerequisite for transposition. Human THAP9 (hTHAP9) is a catalytically active but functionally uncharacterised homologue of DmTNP. ResultsHere we report (using co-IP, pull down, co-localization, PLA) that both the full length as well as truncated hTHAP9 and DmTNP (corresponding to amino-terminal DNA binding and Leucine-rich coiled coil domains) undergo homo-oligomerisation, predominantly in the nuclei of HEK293T cells. Interestingly, the oligomerisation is shown to be partially mediated by DNA. However, mutating the leucines (either individually or together) or deleting the predicted coiled coil region did not significantly affect oligomerisation. We also report that Hcf-1, THAP1, THAP10 and THAP11 are possible protein interaction partners of hTHAP9. ConclusionsThus, we highlight the importance of DNA as well as the amino-terminal regions of both hTHAP9 and DmTNP, for their ability to form higher order oligomeric states. Elucidating the functional relevance of the different putative oligomeric state/s of hTHAP9 would help answer questions about its interaction partners as well as its unknown physiological roles.

scholarly journals Oligomerisation of THAP9 transposase: role of DNA and amino-terminal domains

2020 ◽  
Author(s):  
Hiral M. Sanghavi ◽  
Sharmistha Majumdar
Keyword(s):  
Dna Binding ◽  
Protein Interaction ◽  
Coiled Coil ◽  
Physiological Role ◽  
P Element ◽  
Amino Terminal ◽  
Coiled Coil Region ◽  
Interaction Partners ◽  
Catalytically Active

AbstractActive DNA transposases like the Drosophila P element transposase (DmTNP) undergo oligomerisation as a prerequisite for transposition. Human THAP9 (hTHAP9) is a catalytically active but functionally uncharacterised homologue of DmTNP. Here we report (using co-IP, pull down, co-localization, PLA) that both the full length as well as truncated hTHAP9 and DmTNP (corresponding to amino-terminal DNA binding and Leucine-rich coiled coil domains) undergo homo-oligomerisation, predominantly in the nuclei of HEK293T cells. Interestingly, the oligomerisation is shown to be partially mediated by DNA. However, mutating the leucines (either individually or together) or deleting the predicted coiled coil region did not significantly affect oligomerisation. Thus, we highlight the importance of DNA as well as the amino-terminal regions of both hTHAP9 and DmTNP, for their ability to form higher order oligomeric states. We also report that Hcf-1, THAP1, THAP10 and THAP11 are possible protein interaction partners of hTHAP9. These studies lead to several questions about the different putative oligomeric states of hTHAP9 and how they may be related to its yet unknown physiological role as well as interaction partners.

scholarly journals Megator, an Essential Coiled-Coil Protein that Localizes to the Putative Spindle Matrix during Mitosis inDrosophila

2004 ◽  
Vol 15 (11) ◽  
pp. 4854-4865 ◽  
Author(s):  
Hongying Qi ◽  
Uttama Rath ◽  
Dong Wang ◽  
Ying-Zhi Xu ◽  
Yun Ding ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Matrix Proteins ◽  
P Element ◽  
Structural Basis ◽  
Spindle Matrix ◽  
Terminal Domain ◽  
Coiled Coil Domain ◽  
Coiled Coil Region ◽  
Spherical Structures ◽  
Spindle Structure

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.

scholarly journals X chromosome and autosomal recombination are differentially sensitive to disruptions in SC maintenance

2018 ◽  
Author(s):  
Katherine Kretovich Billmyre ◽  
Cori K. Cahoon ◽  
G. Matthew Heenan ◽  
Emily Wesley ◽  
Zulin Yu ◽  
...  
Keyword(s):  
Protein C ◽  
Coiled Coil ◽  
Double Strand Breaks ◽  
Deletion Mutants ◽  
Strand Breaks ◽  
Homolog Pairing ◽  
Coiled Coil Region ◽  
Or Gene ◽  
Filament Protein

AbstractThe synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double strand breaks (DSBs) into crossovers or gene conversions. The removal of any central region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, we used CRISPR/Cas9 to construct three in-frame deletions within the predicted coiled-coil region of the C(3)G protein. These three deletion mutants disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, allowing us to define the stages of pachytene when the SC is necessary for homolog pairing and recombination. Our studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently than the autosomes.

Targeting of the N-terminal coiled coil oligomerization interface of BCR interferes with the transformation potential of BCR-ABL and increases sensitivity to STI571

Blood ◽  
2003 ◽  
Vol 102 (8) ◽  
pp. 2985-2993 ◽  
Author(s):  
Tim Beissert ◽  
Elena Puccetti ◽  
Andrea Bianchini ◽  
Saskia Güller ◽  
Simone Boehrer ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Coiled Coil ◽  
Chromosome 9 ◽  
Abl Kinase ◽  
Kinase C ◽  
Coiled Coil Region ◽  
Increased Sensitivity ◽  
Transformation Potential

Abstract Translocations involving the abl locus on chromosome 9 fuses the tyrosine kinase c-ABL to proteins harboring oligomerization interfaces such as BCR or TEL, enabling these ABL-fusion proteins (X-ABL) to transform cells and to induce leukemia. The ABL kinase activity is blocked by the ABL kinase inhibitor STI571 which abrogates transformation by X-ABL. To investigate the role of oligomerization for the transformation potential of X-ABL and for the sensitivity to STI571, we constructed ABL chimeras with oligomerization interfaces of proteins involved in leukemia-associated translocations such as BCR, TEL, PML, and PLZF. We assessed the capacity of these chimeras to form high molecular weight (HMW) complexes as compared with p185(BCR-ABL). There was a direct relationship between the size of HMW complexes formed by these chimeras and their capacity to induce factor independence in Ba/F3 cells, whereas there was an inverse relationship between the size of the HMW complexes and the sensitivity to STI571. The targeting of the oligomerization interface of p185(BCR-ABL) by a peptide representing the coiled coil region of BCR reduced its potential to transform fibroblasts and increased sensitivity to STI571. Our results indicate that targeting of the oligomerization interfaces of the X-ABL enhances the effects of STI571 in the treatment of leukemia caused by X-ABL.

scholarly journals Rab11 Function in Trypanosoma brucei: Identification of Conserved and Novel Interaction Partners

2011 ◽  
Vol 10 (8) ◽  
pp. 1082-1094 ◽  
Author(s):  
Carme Gabernet-Castello ◽  
Kelly N. DuBois ◽  
Camus Nimmo ◽  
Mark C. Field
Keyword(s):  
Trypanosoma Brucei ◽  
Intracellular Transport ◽  
Homo Sapiens ◽  
Coiled Coil ◽  
Membrane Composition ◽  
Content Type ◽  
Evolutionarily Conserved ◽  
Interaction Partners ◽  
Hybrid Screening

ABSTRACT The Ras-like GTPase Rab11 is implicated in multiple aspects of intracellular transport, including maintenance of plasma membrane composition and cytokinesis. In metazoans, these functions are mediated in part via coiled-coil Rab11-interacting proteins (FIPs) acting as Rab11 effectors. Additional interaction between Rab11 and the exocyst subunit Sec15 connects Rab11 with exocytosis. We find that FIPs are metazoan specific, suggesting that other factors mediate Rab11 functions in nonmetazoans. We examined Rab11 interactions in Trypanosoma brucei , where endocytosis is well studied and the role of Rab11 in recycling well documented. TbSec15 and TbRab11 interact, demonstrating evolutionary conservation. By yeast two-hybrid screening, we identified additional Rab11 interaction partners. Tb927.5.1640 (designated RBP74) interacted with both Rab11 and Rab5. RBP74 shares a coiled-coil architecture with metazoan FIPs but is unrelated by sequence and appears to play a role in coordinating endocytosis and recycling. A second coiled-coil protein, Tb09.211.4830 (TbAZI1), orthologous to AZI1 in Homo sapiens , interacts exclusively with Rab11. AZI1 is restricted to taxa with motile cilia/flagella. These data suggest that Rab11 functions are mediated by evolutionarily conserved (i.e., AZI1 and Sec15) and potentially lineage-specific (RBP74) interactions essential for the integration of the endomembrane system.

scholarly journals NEMO oligomerization and its ubiquitin-binding properties

2009 ◽  
Vol 421 (2) ◽  
pp. 243-251 ◽  
Author(s):  
Frank J. Ivins ◽  
Mark G. Montgomery ◽  
Susan J. M. Smith ◽  
Aylin C. Morris-Davies ◽  
Ian A. Taylor ◽  
...  
Keyword(s):  
Leucine Zipper ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Coiled Coil ◽  
Nuclear Factor Κb ◽  
Regulatory Subunit ◽  
Activation Process ◽  
Oligomeric State ◽  
Coiled Coil Region ◽  
Ikk Complex

The IKK [IκB (inhibitory κB) kinase] complex is a key regulatory component of NF-κB (nuclear factor κB) activation and is responsible for mediating the degradation of IκB, thereby allowing nuclear translocation of NF-κB and transcription of target genes. NEMO (NF-κB essential modulator), the regulatory subunit of the IKK complex, plays a pivotal role in this process by integrating upstream signals, in particular the recognition of polyubiquitin chains, and relaying these to the activation of IKKα and IKKβ, the catalytic subunits of the IKK complex. The oligomeric state of NEMO is controversial and the mechanism by which it regulates activation of the IKK complex is poorly understood. Using a combination of hydrodynamic techniques we now show that apo-NEMO is a highly elongated, dimeric protein that is in weak equilibrium with a tetrameric assembly. Interaction with peptides derived from IKKβ disrupts formation of the tetrameric NEMO complex, indicating that interaction with IKKα and IKKβ and tetramerization are mutually exclusive. Furthermore, we show that NEMO binds to linear di-ubiquitin with a stoichiometry of one molecule of di-ubiquitin per NEMO dimer. This stoichiometry is preserved in a construct comprising the second coiled-coil region and the leucine zipper and in one that essentially spans the full-length protein. However, our data show that at high di-ubiquitin concentrations a second weaker binding site becomes apparent, implying that two different NEMO–di-ubiquitin complexes are formed during the IKK activation process. We propose that the role of these two complexes is to provide a threshold for activation, thereby ensuring sufficient specificity during NF-κB signalling.

scholarly journals Relevance of Ebola virus VP35 homo-dimerization on the type I interferon cascade inhibition

2019 ◽  
Vol 27 ◽  
pp. 204020661988922 ◽  
Author(s):  
Francesco Di Palma ◽  
Gian Luca Daino ◽  
Venkata Krishnan Ramaswamy ◽  
Angela Corona ◽  
Aldo Frau ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Type I Interferon ◽  
Coiled Coil ◽  
Coarse Grained ◽  
Type I ◽  
Wild Type ◽  
Terminal Domain ◽  
Coiled Coil Region ◽  
Interferon Antagonism

Ebola virus high lethality relies on its ability to efficiently bypass the host innate antiviral response, which senses the viral dsRNA through the RIG-I receptor and induces type I interferon α/β production. In the bypassing action, the Ebola virus protein VP35 plays a pivotal role at multiple levels of the RIG-I cascade, masking the viral 5′-triphosphorylated dsRNA from RIG-I, and interacting with other cascade components. The VP35 type I interferon inhibition is exerted by the C-terminal domain, while the N-terminal domain, containing a coiled-coil region, is primarily required for oligomerization. However, mutations at key VP35 residues L90/93/107A (VP35-3m) in the coiled-coil region were reported to affect oligomerization and reduce type I interferon antagonism, indicating a possible but unclear role of homo-oligomerization on VP35 interaction with the RIG-I pathway components. In this work, we investigated the VP35 dimerization thermodynamics and its contribution to type I interferon antagonism by computational and biological methods. Focusing on the coiled-coil region, we combined coarse-grained and all-atom simulations on wild type VP35 and VP35-3m homo-dimerization. According to our results, wild type VP35 coiled-coil is able to self-assemble into dimers, while VP35-3m coiled-coil shows poor propensity to even dimerize. Free-energy calculations confirmed the key role of L90, L93 and L107 in stabilizing the coiled-coil homo-dimeric structure. In vitro type I interferon antagonism studies, using full-length wild type VP35 and VP35-3m, revealed that VP35 homo-dimerization is an essential preliminary step for dsRNA binding, which appears to be the main factor of the VP35 RIG-I cascade inhibition, while it is not essential to block the other steps.

scholarly journals Structural Basis of Mitochondrial Scaffolds by Prohibitin Complexes: Insight into a Role of the Coiled-Coil Region

iScience ◽  
2019 ◽  
Vol 19 ◽  
pp. 1065-1078 ◽  
Author(s):  
Takahiro Yoshinaka ◽  
Hidetaka Kosako ◽  
Takuma Yoshizumi ◽  
Ryo Furukawa ◽  
Yu Hirano ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Structural Basis ◽  
Coiled Coil Region ◽  
Insight Into

scholarly journals X chromosome and autosomal recombination are differentially sensitive to disruptions in SC maintenance

2019 ◽  
Vol 116 (43) ◽  
pp. 21641-21650 ◽  
Author(s):  
Katherine Kretovich Billmyre ◽  
Cori K. Cahoon ◽  
G. Matthew Heenan ◽  
Emily R. Wesley ◽  
Zulin Yu ◽  
...  
Keyword(s):  
Protein C ◽  
Coiled Coil ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Deletion Mutations ◽  
Homolog Pairing ◽  
Coiled Coil Region ◽  
Or Gene ◽  
Filament Protein

The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double-strand breaks (DSBs) into crossovers or gene conversions. The removal of any central-region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, we used CRISPR/Cas9 to construct 3 in-frame deletions within the predicted coiled-coil region of the C(3)G protein. Since these 3 deletion mutations disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, they allow us to define the stages of pachytene when the SC is necessary for homolog pairing and recombination during pachytene. Our studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently from the autosomes.

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