Identification of bZIP Interaction Partners of Viral Proteins HBZ, MEQ, BZLF1, and K-bZIP Using Coiled-Coil Arrays

Human ATG8 family proteins (ATG8s) are active in all steps of the macroautophagy pathway, and their lipidation is essential for autophagosome formation. Lipidated ATG8s anchored to the outer surface of the phagophore serve as scaffolds for binding of other core autophagy proteins and various effector proteins involved in trafficking or fusion events, whereas those at the inner surface are needed for assembly of selective autophagy substrates. Their scaffolding role depends on specific interactions between the LC3-interacting region (LIR) docking site (LDS) in ATG8s and LIR motifs in various interaction partners. LC3B is phosphorylated at Thr-50 within the LDS by serine/threonine kinase (STK) 3 and STK4. Here, we identified LIR motifs in STK3 and atypical protein kinase Cζ (PKCζ) and never in mitosis A (NIMA)-related kinase 9 (NEK9). All three kinases phosphorylated LC3B Thr-50 in vitro. A phospho-mimicking substitution of Thr-50 impaired binding of several LIR-containing proteins, such as ATG4B, FYVE, and coiled-coil domain-containing 1 (FYCO1), and autophagy cargo receptors p62/sequestosome 1 (SQSTM1) and neighbor of BRCA1 gene (NBR1). NEK9 knockdown or knockout enhanced degradation of the autophagy receptor and substrate p62. Of note, the suppression of p62 degradation was mediated by NEK9-mediated phosphorylation of LC3B Thr-50. Consistently, reconstitution of LC3B-KO cells with the phospho-mimicking T50E variant inhibited autophagic p62 degradation. PKCζ knockdown did not affect autophagic p62 degradation, whereas STK3/4 knockouts inhibited autophagic p62 degradation independently of LC3B Thr-50 phosphorylation. Our findings suggest that NEK9 suppresses LC3B-mediated autophagy of p62 by phosphorylating Thr-50 within the LDS of LC3B.

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

Eukaryotic Cell ◽

10.1128/ec.05098-11 ◽

2011 ◽

Vol 10 (8) ◽

pp. 1082-1094 ◽

Cited By ~ 13

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.

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Towards identifying preferred interaction partners of fluorinated amino acids within the hydrophobic environment of a dimeric coiled coil peptide

Organic & Biomolecular Chemistry ◽

10.1039/b917205j ◽

2010 ◽

Vol 8 (6) ◽

pp. 1382 ◽

Cited By ~ 24

Author(s):

Toni Vagt ◽

Elisabeth Nyakatura ◽

Mario Salwiczek ◽

Christian Jäckel ◽

Beate Koksch

Keyword(s):

Amino Acids ◽

Coiled Coil ◽

Fluorinated Amino Acids ◽

Interaction Partners

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Comprehensive Identification of Human bZIP Interactions with Coiled-Coil Arrays

Science ◽

10.1126/science.1084648 ◽

2003 ◽

Vol 300 (5628) ◽

pp. 2097-2101 ◽

Cited By ~ 311

Author(s):

J. R. S. Newman

Keyword(s):

Coiled Coil ◽

Coil Arrays

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

10.21203/rs.3.rs-100025/v1 ◽

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.

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

10.1101/2020.10.06.328815 ◽

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.

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Faculty Opinions recommendation of Comprehensive identification of human bZIP interactions with coiled-coil arrays.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014173.192950 ◽

2003 ◽

Author(s):

Deyou Zheng

Keyword(s):

Coiled Coil ◽

Coil Arrays

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Analysis of the Putative Nucleoporin POM33 in the Filamentous Fungus Sordaria macrospora

Journal of Fungi ◽

10.3390/jof7090682 ◽

2021 ◽

Vol 7 (9) ◽

pp. 682

Author(s):

Anika Groth ◽

Kerstin Schmitt ◽

Oliver Valerius ◽

Britta Herzog ◽

Stefanie Pöggeler

Keyword(s):

Filamentous Fungus ◽

Vegetative Growth ◽

Protein Translocation ◽

Coiled Coil ◽

End Joining ◽

Hyphal Fusion ◽

Sordaria Macrospora ◽

Body Development ◽

Interaction Partners ◽

Non Homologous End Joining

In the filamentous fungus Sordaria macrospora (Sm), the STRIPAK complex is required for vegetative growth, fruiting-body development and hyphal fusion. The SmSTRIPAK core consists of the striatin homolog PRO11, the scaffolding subunit of phosphatase PP2A, SmPP2AA, and its catalytic subunit SmPP2Ac1. Among other STRIPAK proteins, the recently identified coiled-coil protein SCI1 was demonstrated to co-localize around the nucleus. Pulldown experiments with SCI identified the transmembrane nucleoporin (TM Nup) SmPOM33 as a potential nuclear-anchor of SmSTRIPAK. Localization studies revealed that SmPOM33 partially localizes to the nuclear envelope (NE), but mainly to the endoplasmic reticulum (ER). We succeeded to generate a ∆pom33 deletion mutant by homologous recombination in a new S. macrospora Δku80 recipient strain, which is defective in non-homologous end joining. Deletion of Smpom33 did neither impair vegetative growth nor sexual development. In pulldown experiments of SmPOM33 followed by LC/MS analysis, ER-membrane proteins involved in ER morphology, protein translocation, glycosylation, sterol biosynthesis and Ca2+-transport were significantly enriched. Data are available via ProteomeXchange with identifier PXD026253. Although no SmSTRIPAK components were identified as putative interaction partners, it cannot be excluded that SmPOM33 is involved in temporarily anchoring the SmSTRIPAK to the NE or other sites in the cell.

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TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

Biochemical Journal ◽

10.1042/bcj20190359 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3241-3260

Author(s):

Sindhu Wisesa ◽

Yasunori Yamamoto ◽

Toshiaki Sakisaka

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Membrane Protein ◽

Mammalian Cells ◽

Coiled Coil ◽

Transmembrane Domains ◽

Domain Family ◽

Coiled Coil Domain ◽

U2os Cells ◽

Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

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