FRET ‐based analysis and molecular modeling of the human GPN ‐loop GTP ases 1 and 3 heterodimer unveils a dominant‐negative protein complex

FEBS Journal ◽  
2019 ◽  
Vol 286 (23) ◽  
pp. 4797-4818 ◽  
Author(s):  
Gema R. Cristóbal‐Mondragón ◽  
Bárbara Lara‐Chacón ◽  
Ángel Santiago ◽  
Víctor De‐la‐Rosa ◽  
Rogelio González‐González ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Protein Complex ◽  
Dominant Negative

scholarly journals Atypical Protein Kinase C Is Involved in the Evolutionarily Conserved Par Protein Complex and Plays a Critical Role in Establishing Epithelia-Specific Junctional Structures

2001 ◽  
Vol 152 (6) ◽  
pp. 1183-1196 ◽  
Author(s):  
Atsushi Suzuki ◽  
Tomoyuki Yamanaka ◽  
Tomonori Hirose ◽  
Naoyuki Manabe ◽  
Keiko Mizuno ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Polarity ◽  
Protein Complex ◽  
Mdck Cells ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Surface Polarity ◽  
C Elegans ◽  
Evolutionarily Conserved

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

scholarly journals Spc24 and Stu2 Promote Spindle Integrity When DNA Replication Is Stalled

2007 ◽  
Vol 18 (8) ◽  
pp. 2805-2816 ◽  
Author(s):  
Lina Ma ◽  
Jennifer McQueen ◽  
Lara Cuschieri ◽  
Jackie Vogel ◽  
Vivien Measday
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Protein Complex ◽  
Normal Cell ◽  
Budding Yeast ◽  
S Phase ◽  
Dominant Negative ◽  
Truncated Form ◽  
Spindle Elongation ◽  
Normal Cell Cycle

The kinetochore, a protein complex that links chromosomes to microtubules (MTs), is required to prevent spindle expansion during S phase in budding yeast, but the mechanism of how the kinetochore maintains integrity of the bipolar spindle before mitosis is not well understood. Here, we demonstrate that a mutation of Spc24, a component of the conserved Ndc80 kinetochore complex, causes lethality when cells are exposed to the DNA replication inhibitor hydroxyurea (HU) due to premature spindle expansion and segregation of incompletely replicated DNA. Overexpression of Stu1, a CLASP-related MT-associated protein or a truncated form of the XMAP215 orthologue Stu2 rescues spc24-9 HU lethality and prevents spindle expansion. Truncated Stu2 likely acts in a dominant-negative manner, because overexpression of full-length STU2 does not rescue spc24-9 HU lethality, and spindle expansion in spc24-9 HU-treated cells requires active Stu2. Stu1 and Stu2 localize to the kinetochore early in the cell cycle and Stu2 kinetochore localization depends on Spc24. We propose that mislocalization of Stu2 results in premature spindle expansion in S phase stalled spc24-9 mutants. Identifying factors that restrain spindle expansion upon inhibition of DNA replication is likely applicable to the mechanism by which spindle elongation is regulated during a normal cell cycle.

scholarly journals Characterization of an apical ceramide-enriched compartment regulating ciliogenesis

2012 ◽  
Vol 23 (16) ◽  
pp. 3156-3166 ◽  
Author(s):  
Qian He ◽  
Guanghu Wang ◽  
Somsankar Dasgupta ◽  
Michael Dinkins ◽  
Gu Zhu ◽  
...  
Keyword(s):  
Protein Complex ◽  
Primary Cilia ◽  
Mdck Cells ◽  
Trichostatin A ◽  
Acid Sphingomyelinase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Tubulin Acetylation ◽  
Magnetic Sorting

We show that in Madin–Darby canine kidney (MDCK) cells, an apical ceramide-enriched compartment (ACEC) at the base of primary cilia is colocalized with Rab11a. Ceramide and Rab11a vesicles isolated by magnetic sorting contain a highly similar profile of proteins (atypical protein kinase C [aPKC], Cdc42, Sec8, Rab11a, and Rab8) and ceramide species, suggesting the presence of a ciliogenic protein complex associated with ceramide at the ACEC. It is intriguing that C16 and C18 ceramide, although less abundant ceramide species in MDCK cells, are highly enriched in ceramide and Rab11a vesicles. Expression of a ceramide-binding but dominant-negative mutant of aPKC suppresses ciliogenesis, indicating that the association of ceramide with aPKC is critical for the formation of this complex. Our results indicate that ciliogenic ceramide is derived from apical sphingomyelin (SM) that is endocytosed and then converted to the ACEC. Consistently, inhibition of acid sphingomyelinase with imipramine disrupts ACEC formation, association of ciliogenic proteins with Rab11a vesicles, and cilium formation. Ciliogenesis is rescued by the histone deacetylase (HDAC) inhibitor trichostatin A, indicating that ceramide promotes tubulin acetylation in cilia. Taken together, our results suggest that the ACEC is a novel compartment in which SM-derived ceramide induces formation of a ciliogenic lipid–protein complex that sustains primary cilia by preventing deacetylation of microtubules.

scholarly journals Integrin-linked Kinase Interactions with ELMO2 Modulate Cell Polarity

2009 ◽  
Vol 20 (13) ◽  
pp. 3033-3043 ◽  
Author(s):  
Ernest Ho ◽  
Tames Irvine ◽  
Gregory J.A. Vilk ◽  
Gilles Lajoie ◽  
Kodi S. Ravichandran ◽  
...  
Keyword(s):  
Cell Motility ◽  
Cell Polarity ◽  
Tissue Regeneration ◽  
Protein Complex ◽  
Cell Polarization ◽  
Dominant Negative ◽  
Forward Movement ◽  
Directed Migration ◽  
Polarized Cells ◽  
Integrin Linked Kinase

Cell polarization is a key prerequisite for directed migration during development, tissue regeneration, and metastasis. Integrin-linked kinase (ILK) is a scaffold protein essential for cell polarization, but very little is known about the precise mechanisms whereby ILK modulates polarization in normal epithelia. Elucidating these mechanisms is essential to understand tissue morphogenesis, transformation, and repair. Here we identify a novel ILK protein complex that includes Engulfment and Cell Motility 2 (ELMO2). We also demonstrate the presence of RhoG in ILK–ELMO2 complexes, and the localization of this multiprotein species specifically to the leading lamellipodia of polarized cells. Significantly, the ability of RhoG to bind ELMO is crucial for ILK induction of cell polarization, and the joint expression of ILK and ELMO2 synergistically promotes the induction of front-rear polarity and haptotactic migration. This places RhoG–ELMO2–ILK complexes in a key position for the development of cell polarity and forward movement. Although ILK is a component of many diverse multiprotein species that may contribute to cell polarization, expression of dominant-negative ELMO2 mutants is sufficient to abolish the ability of ILK to promote cell polarization. Thus, its interaction with ELMO2 and RhoG is essential for the ability of ILK to induce front-rear cell polarity.

Identification of a Novel Acetyl-CoA Binding Protein (EST1) Which Epigenetically Regulates the Expression of Erythroid-Specific Genes

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4761-4761
Author(s):  
Tohru Fujiwara ◽  
Tsuyoshi Ikura ◽  
Satoshi Ichikawa ◽  
Shinichiro Takahashi ◽  
Kazumichi Furuyama ◽  
...  
Keyword(s):  
Western Blot ◽  
Protein Complex ◽  
Es Cells ◽  
Differential Expression Analysis ◽  
Erythroid Differentiation ◽  
Dominant Negative ◽  
Erythroid Cells ◽  
Wild Type ◽  
Acetyl Coa ◽  
In Erythroid Cells

Abstract (Introduction) Embryonic stem (ES) cells that lack 5-aminolevulinate synthase-2 (ALAS2) gene provide a valuable model in dissecting molecular events in which heme is required during erythroid differentiation. Recently, we identified a novel acetyltransferase-like gene (EST1) through differential expression analysis between wild-type and heme-deficient erythroblasts as a potential downstream target gene of heme (BBRC2006;340:105–110, ASH 2007). EST1 belongs to the GNAT (GCN5-related N-acetyltransferase) superfamily since it contains the highly conserved amino acid residues (motif A), known as acetyl-CoA binding domain. Since the GNAT superfamily contains a wide variety of acetyltransferases with different substrates or with unknown functions, probing how EST1 might be required for biological events in erythroid cells, could be informative. Here, we investigated the role of EST1 during erythroid differentiation. (Methods) EST1 was constitutively expressed using Flag/HA-tagged retroviral vector into mouse erythroleukemia (MEL) cell line. EST1 protein complex was purified by affinity chromatography from nuclear extract of EST1-expessed MEL cells. To obtain dominant-negative EST1-expressing cells, both Arg-62 and Gly-65 within EST1 were substituted to glutamic acid, and similarly transduced into MEL cells. These mutations have been widely applied for abolishment of acetyl-CoA binding activity. For depletion of endogenous EST1, siRNAs specific for EST1 were introduced into Hepa1c1c7 cells (Results) Although recombinant EST1 protein did not have acetylase activity for free histones in vitro (ASH 2007), EST1 protein forms a multimeric protein complex. Western blot analysis using FLAG-eluted polypeptides revealed the presence of GCN5, TRRAP, SPT3 and GATA-1, implying that this protein complex might participate in the transcriptional regulation of erythroid-specific genes. Following EST1 depletion in Hepa1c1c cells, a significant decrease in the acetylation of H3 and a mild decrease in that of H4, were observed by Western blot analyses. Similarly, a significant decrease in the acetylation of H3 and a mild decrease in that of H4 were also observed in dominant-negative than in wild-type EST1-expressing MEL cells. Furthermore, the level of bmajor and ALAS2 mRNA were significantly lower in dominant-negative than in wild-type EST1-expressing MEL cells upon treatment with 1.5% DMSO for both 48h and 72h. We are currently exploring mechanisms of how EST1 participates in the regulation of histone modification in erythroid cells. (Conclusion) EST1 may epigenetically regulate a subset of erythroid-specific genes under control of heme. Further elucidation of the function of the EST1 gene would enhance our understanding of the transcriptional network involving erythroid differentiation.

Consequences of intramolecular dityrosine formation on a DNA–protein complex: a molecular modeling study

2005 ◽  
Vol 72 (2-3) ◽  
pp. 271-278 ◽  
Author(s):  
Julien Gras ◽  
Denise Sy ◽  
Séverine Eon ◽  
Michel Charlier ◽  
Melanie Spotheim-Maurizot
Keyword(s):  
Molecular Modeling ◽  
Protein Complex ◽  
Molecular Modeling Study ◽  
Modeling Study

scholarly journals Transcription of the dominant-negative helix-loop-helix protein Id1 is regulated by a protein complex containing the immediate-early response gene Egr-1.

1996 ◽  
Vol 16 (5) ◽  
pp. 2418-2430 ◽  
Author(s):  
O Tournay ◽  
R Benezra
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Protein Complex ◽  
Early Response ◽  
Dominant Negative ◽  
Upstream Region ◽  
Response Gene ◽  
Enhancer Element ◽  
Early Response Gene ◽  
Helix Loop Helix

The expression of Id1, a helix-loop-helix protein which inhibits the activity of basic helix-loop-helix transcription factors, is down-regulated during cellular differentiation and cell cycle withdrawal both in tissue culture models and in mouse embryos. In order to study the mechanism of control of Idl expression, we have isolated a 210-bp enhancer element in the upstream region of the Id1 gene whose activity recapitulates Id1 expression in C2C12 muscle cells and C3H10T1/2 fibroblasts: i.e., this element is active in proliferating cells in the presence of serum and completely inactivated upon mitogen depletion, cell cycle withdrawal, and (in the case of C2C12) induced myoblast differentiation. Using linker-scanning mutations and site-directed mutagenesis in transient transfection experiments, we have identified two functional elements within the 210-bp enhancer which are required for proper serum responsiveness. One element (A) contains a consensus Egr-1 binding site and additional flanking sequences required for optimal activity, and the other element (B) fits no known consensus. Gel shift experiments demonstrate that the protein complex binding to the A site contains Egr-1 and other proteins. This complex as well as a protein complex that binds to the B site is lost within 24 h of serum depletion, correlating with the down-regulation of Id1 expression. On the basis of these findings, we propose that the regulation of the Id1 response to serum is mediated in part by the early response gene Egr-1 and as such provides a signaling link between the early-growth-response transcription factors and dominant-negative helix-loop-helix proteins.

High-Resolution electron crystallography of the light-harvesting chlorophyll-a/b protein complex from chloroplast membranes

1990 ◽  
Vol 48 (1) ◽  
pp. 610-610
Author(s):  
Werner Kühlbrandt ◽  
Da Neng Wang ◽  
K.H. Downing
Keyword(s):  
High Resolution ◽  
Electron Diffraction ◽  
Chlorophyll A ◽  
Protein Complex ◽  
Structural Integrity ◽  
Light Harvesting ◽  
Lhc Ii ◽  
Diffraction Patterns ◽  
Difference Fourier ◽  
2D Crystals

The light-harvesting chlorophyll-a/b protein complex (LHC-II) is the most abundant membrane protein in the chloroplasts of green plants where it functions as a molecular antenna of solar energy for photosynthesis. We have grown two-dimensional (2d) crystals of the purified, detergent-solubilized LHC-II . The crystals which measured 5 to 10 μm in diameter were stabilized for electron microscopy by washing with a 0.5% solution of tannin. Electron diffraction patterns of untilted 2d crystals cooled to 130 K showed sharp spots to 3.1 Å resolution. Spot-scan images of 2d crystals were recorded at 160 K with the Berkeley microscope . Images of untilted crystals were processed, using the unbending procedure by Henderson et al . A projection map of the complex at 3.7Å resolution was generated from electron diffraction amplitudes and high-resolution phases obtained by image processing .A difference Fourier analysis with the same image phases and electron diffraction amplitudes recorded of frozen, hydrated specimens showed no significant differences in the 3.7Å projection map. Our tannin treatment therefore does not affect the structural integrity of the complex.

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