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 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 The Centrosomal, Putative Tumor Suppressor Protein TACC2 Is Dispensable for Normal Development, and Deficiency Does Not Lead to Cancer

2004 ◽  
Vol 24 (14) ◽  
pp. 6403-6409 ◽  
Author(s):  
Michael M. Schuendeln ◽  
Roland P. Piekorz ◽  
Christian Wichmann ◽  
Youngsoo Lee ◽  
Peter J. McKinnon ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Cycle Progression ◽  
Tumor Development ◽  
Coiled Coil ◽  
Physiological Role ◽  
Tumor Suppressor Protein ◽  
Wild Type ◽  
Mouse Development

ABSTRACT TACC2 is a member of the transforming acidic coiled-coil-containing protein family and is associated with the centrosome-spindle apparatus during cell cycling. In vivo, the TACC2 gene is expressed in various splice forms predominantly in postmitotic tissues, including heart, muscle, kidney, and brain. Studies of human breast cancer samples and cell lines suggest a putative role of TACC2 as a tumor suppressor protein. To analyze the physiological role of TACC2, we generated mice lacking TACC2. TACC2-deficient mice are viable, develop normally, are fertile, and lack phenotypic changes compared to wild-type mice. Furthermore, TACC2 deficiency does not lead to an increased incidence of tumor development. Finally, in TACC2-deficient embryonic fibroblasts, proliferation and cell cycle progression as well as centrosome numbers are comparable to those in wild-type cells. Therefore, TACC2 is not required, nonredundantly, for mouse development and normal cell proliferation and is not a tumor suppressor protein.

The CBFb-SMMHC Oncoprotein Inhibits Binding of the Runx1 Runt Domain to DNA.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2709-2709
Author(s):  
John H. Bushweller ◽  
Stephen M. Lukasik ◽  
Nancy A. Speck
Keyword(s):  
Dna Binding ◽  
Fetal Liver ◽  
Chromosomal Rearrangements ◽  
Coiled Coil ◽  
Embryonic Lethality ◽  
Runt Domain ◽  
Viable Approach ◽  
Muscle Myosin ◽  
Silencer Element

Abstract Core-binding factors (CBFs) are heterodimeric transcriptional factors consisting of a DNA-binding Runx1 (CBFα) subunit and a CBFβ subunit. Cbfβ allosterically increases the affinity of Runx1 for DNA ~2.5 fold. CBF subunits are encoded by four genes in mammals. RUNX1 (AML1), RUNX2, and RUNX3 encode for CBFα subunits, and CBFB encodes the CBFβ subunit. Homozygous disruption of either the Runx1 or the Cbfb genes in mice results in essentially identical phenotypes: midgestation embryonic lethality accompanied by extensive hemorrhaging and a profound block at the fetal liver stage of hematopoiesis. In humans, chromosomal rearrangements that disrupt the Runx1 and CBFB genes are associated with a significant percentage of leukemias. CBFβ is disrupted in acute myeloid leukemia by inv(16)(p13;q22), t(16;16), and del(16)(q22). These translocations result in the production of novel fusion proteins containing most of the CBFβ protein fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (SMMHC) encoded by the MYH11 gene. A knock-in of the CBFB-MYH11 allele in mice resulted in embryonic lethality with a profound block in hematopoietic development, the same phenotype observed for the Runx1 and Cbfb knockouts. We recently demonstrated that the CBFβ-SMMHC fusion protein binds to the DNA binding Runt domain from Runx1 with both higher affinity and altered stoichiometry relative to native CBFβ. We also provided NMR-based evidence for multiple sites of contact between Runx1 and CBFβ-SMMHC, proving the role of the SMMHC sequence in creating this altered affinity. Here we demonstrate that CBFβ-SMMHC inhibits DNA binding of the Runx1 Runt domain by ~6-fold for the CD4 dual-site silencer element. Cross-saturation NMR mapping on the Runt domain in complex with CBFβ-SMMHC reveals that the SMMHC portion of the oncoprotein makes contacts with β-strands 1 and 2 in the Runt domain. We propose that the inhibition of DNA-binding and increased affinity combine to mediate the dysregulation of Runx-regulated genes caused by CBFβ-SMMHC. These results also clearly suggest that targeting of the CBFβ-SMMHC protein for drug development may well be a viable approach for the treatment of the associated leukemia.

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.

scholarly journals Sharp kinking of a coiled-coil in MutS allows DNA binding and release

2019 ◽  
Vol 47 (16) ◽  
pp. 8888-8898 ◽  
Author(s):  
Doreth Bhairosing-Kok ◽  
Flora S Groothuizen ◽  
Alexander Fish ◽  
Shreya Dharadhar ◽  
Herrie H K Winterwerp ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Dna Binding ◽  
Mismatch Repair ◽  
Coiled Coil ◽  
Site Specific ◽  
Dna Mismatch ◽  
Hinge Point ◽  
Coiled Coil Region ◽  
Kink Site

Abstract DNA mismatch repair (MMR) corrects mismatches, small insertions and deletions in DNA during DNA replication. While scanning for mismatches, dimers of MutS embrace the DNA helix with their lever and clamp domains. Previous studies indicated generic flexibility of the lever and clamp domains of MutS prior to DNA binding, but whether this was important for MutS function was unknown. Here, we present a novel crystal structure of DNA-free Escherichia coli MutS. In this apo-structure, the clamp domains are repositioned due to kinking at specific sites in the coiled-coil region in the lever domains, suggesting a defined hinge point. We made mutations at the coiled-coil hinge point. The mutants made to disrupt the helical fold at the kink site diminish DNA binding, whereas those made to increase stability of coiled-coil result in stronger DNA binding. These data suggest that the site-specific kinking of the coiled-coil in the lever domain is important for loading of this ABC-ATPase on DNA.

Structure and function of asterosaps, sperm-activating peptides from the jelly coat of starfish eggs

Zygote ◽  
1996 ◽  
Vol 4 (3) ◽  
pp. 237-245 ◽  
Author(s):  
Takuya Nishigaki ◽  
Kazuyoshi Chiba ◽  
Wataru Miki ◽  
Motonori Hoshi
Keyword(s):  
Acrosome Reaction ◽  
Physiological Role ◽  
Structural Diversity ◽  
Asterias Amurensis ◽  
Amino Terminal ◽  
Jelly Coat ◽  
Ionisation Mass Spectrometry ◽  
Egg Jelly ◽  
And Function

SummaryJelly coat of starfish eggs has the capacity to activate homologous spermatozoa and induce the acrosome reaction. We have isolated 12 sperm-activating peptides (SAPs) from the egg jelly of the starfish, Asterias amurensis. Eleven SAPs were structurally identified by sequence analysis and electro-spray ionisation mass spectrometry. All of them are glutamine-rich tetratriacontapeptides with an intramolecular disulphide linkage between Cys8 and Cys32. They are much larger than sea urchin SAPs and do not show any significant sequence similarities to known proteins. Thus we have collectively named them asterosaps. The amino terminal region, where structural diversity of asterosaps is observed, is not important for their activity, whereas the disulphide linkage is essential. Asterosaps do not induce the acrosome reaction by themselves, but are able to induce the acrosome reaction in combination with an egg jelly glycoconjugate named ARIS. Furthermore, anti-asterosap rabbit antibody significantly decreased the acrosome reaction-inducing activity of the jelly solution and the activity was restored by addition of excess asterosap. These results support our hypothesis that the main physiological role of SAPs is the induction of the acrosome reaction in cooperation with two other jelly components, ARIS and Co-ARIS.

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.

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