Proteasomal interactors control activities as diverse as the cell cycle and glutaminergic neurotransmission

2003 ◽  
Vol 31 (2) ◽  
pp. 470-473 ◽  
Author(s):  
K. Rezvani ◽  
M. Mee ◽  
S. Dawson ◽  
J. McIlhinney ◽  
J. Fujita ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Metabotropic Glutamate Receptors ◽  
Carcinoma Cells ◽  
Genetic Screens ◽  
Yeast Two Hybrid ◽  
Metabotropic Glutamate ◽  
Biological Studies ◽  
E2f Transcription Factor ◽  
Biochemical Analyses ◽  
Two Hybrid

The six regulatory non-redundant ATPases in the base of the 19 S regulator of the 26 S proteasome belong to the AAA superfamily of ATPases. Yeast two-hybrid genetic screens, biochemical analyses and cell biological studies have identified and characterized new interactors of the human S6 (rpt3) and S8 (rpt6) ATPases of the 19 S regulator of the 26 S proteasome. The S6 ATPase interacts with gankyrin. This protein is found in purified human 26 S proteasomes and in a smaller complex(es) containing CDK4 and free S6 ATPase. Gankyrin overexpression causes the phosphorylation of the retinoblastoma protein (pRb) and the release of E2F transcription factor to trigger the expression of DNA synthesis genes. Gankyrin is oncogenic in nude mice and is overexpressed in hepatocellular carcinoma cells (HCCs). The S8 ATPase interacts with members of the large Homer-3 protein family. There are three Homer genes; the Homer 1 and 2 gene products control trafficking and calcium-store-related functions of metabotropic glutamate receptors (e.g. mGluR1α). Homer-3A11 by binding to the S8 ATPase brings mGluR1α to the 26 S proteasome for degradation. The degradation of mGluR1α is blocked by proteasomal inhibitors and by overexpression of the N-terminus of Homer which binds to the receptor. The S8 ATPase and mGluR1α are co-localized in Purkinje dendrites in rat cerebellum. The data are discussed in terms of the regulation of the cell cycle and glutaminergic receptor functions by the 26 S proteasome.

scholarly journals Ang2/Fat-Free Is a Conserved Subunit of the Golgi-associated Retrograde Protein Complex

2010 ◽  
Vol 21 (19) ◽  
pp. 3386-3395 ◽  
Author(s):  
F. Javier Pérez-Victoria ◽  
Christina Schindler ◽  
Javier G. Magadán ◽  
Gonzalo A. Mardones ◽  
Cédric Delevoye ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Coiled Coil ◽  
Yeast Two Hybrid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Coiled Coil Region ◽  
Significant Homology ◽  
Biochemical Analyses ◽  
Golgi Network ◽  
Two Hybrid ◽  
Garp Complex

The Golgi-associated retrograde protein (GARP) complex mediates tethering and fusion of endosome-derived transport carriers to the trans-Golgi network (TGN). In the yeast Saccharomyces cerevisiae, GARP comprises four subunits named Vps51p, Vps52p, Vps53p, and Vps54p. Orthologues of the GARP subunits, except for Vps51p, have been identified in all other eukaryotes. A yeast two-hybrid screen of a human cDNA library yielded a phylogenetically conserved protein, Ang2/Fat-free, which interacts with human Vps52, Vps53 and Vps54. Human Ang2 is larger than yeast Vps51p, but exhibits significant homology in an N-terminal coiled-coil region that mediates assembly with other GARP subunits. Biochemical analyses show that human Ang2, Vps52, Vps53 and Vps54 form an obligatory 1:1:1:1 complex that strongly interacts with the regulatory Habc domain of the TGN SNARE, Syntaxin 6. Depletion of Ang2 or the GARP subunits similarly impairs protein retrieval to the TGN, lysosomal enzyme sorting, endosomal cholesterol traffic¤ and autophagy. These findings indicate that Ang2 is the missing component of the GARP complex in most eukaryotes.

scholarly journals Transcription Activator Swi6 Interacts with Mbp1 in MluI Cell Cycle Box-Binding Complex and Regulates Hyphal Differentiation and Virulence in Beauveria bassiana

2021 ◽  
Vol 7 (6) ◽  
pp. 411
Author(s):  
Jin-Li Ding ◽  
Jia Hou ◽  
Xiu-Hui Li ◽  
Ming-Guang Feng ◽  
Sheng-Hua Ying
Keyword(s):  
Cell Cycle ◽  
Direct Interaction ◽  
Hyphal Growth ◽  
Transcription Activator ◽  
Liquid Media ◽  
Yeast Two Hybrid ◽  
Genetic Pathways ◽  
Morphological Transitions ◽  
Lethal Time ◽  
Two Hybrid

Mbp1 protein acts as a DNA-binding protein in MluI cell cycle box-binding complex (MBF) and plays an essential role in filamentous myco-pathogen Beauveria bassiana.In the current study, BbSwi6 (a homologue of yeast Swi6) was functionally characterized in B.bassiana. Both BbSwi6 and BbMbp1 localize in the nucleus and display a direct interaction relationship which is indicated by a yeast two-hybrid assay. BbSwi6 significantly contributes to hyphal growth, asexual sporulation and virulence. On the aerial surface, ΔBbSwi6 grew slower on various nutrients and displayed abnormal conidia-producing structures, which hardly produced conidia. In liquid media, BbSwi6 loss led to 90% reduction in blastospore yield. Finally, the virulence of the ΔBbSwi6 mutant was modestly weakened with a reduction of 20% in median lethal time. Comparative transcriptomics revealed that BbSwi6 mediated different transcriptomes during fungal development into conidia and blastospores. Notably, under the indicated condition, the BbSwi6-mediated transcriptome significantly differed to that mediated by BbMbp1. Our results demonstrate that, in addition to their roles as the interactive components in MBF, BbSwi6 and BbMbp1 mediate divergent genetic pathways during morphological transitions in B. bassiana.

scholarly journals Direct interaction of β-dystroglycan with F-actin

2003 ◽  
Vol 375 (2) ◽  
pp. 329-337 ◽  
Author(s):  
Yun-Ju CHEN ◽  
Heather J. SPENCE ◽  
Jacqueline M. CAMERON ◽  
Thomas JESS ◽  
Jane L. ILSLEY ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Direct Interaction ◽  
Cytoplasmic Tail ◽  
Yeast Two Hybrid ◽  
Glycoprotein Complex ◽  
Biological Studies ◽  
Dystrophin Glycoprotein Complex ◽  
Dystrophin Glycoprotein ◽  
Two Hybrid ◽  
Sarcolemmal Integrity

Dystroglycans are essential transmembrane adhesion receptors for laminin. α-Dystroglycan is a highly glycosylated extracellular protein that interacts with laminin in the extracellular matrix and the transmembrane region of β-dystroglycan. β-Dystroglycan, via its cytoplasmic tail, interacts with dystrophin and utrophin and also with the actin cytoskeleton. As a part of the dystrophin–glycoprotein complex of muscles, dystroglycan is also important in maintaining sarcolemmal integrity. Mutations in dystrophin that lead to Duchenne muscular dystrophy also lead to a loss of dystroglycan from the sarcolemma, and chimaeric mice lacking muscle dystroglycan exhibit a severe muscular dystrophy phenotype. Using yeast two-hybrid analysis and biochemical and cell biological studies, we show, in the present study, that the cytoplasmic tail of β-dystroglycan interacts directly with F-actin and, furthermore, that it bundles actin filaments and induces an aberrant actin phenotype when overexpressed in cells.

Determining calmodulin binding to metabotropic glutamate receptors with distinct protein-interaction methods

2004 ◽  
Vol 32 (5) ◽  
pp. 868-870 ◽  
Author(s):  
K. Lidwell ◽  
J. Dillon ◽  
A. Sihota ◽  
V. O'Connor ◽  
B. Pilkington
Keyword(s):  
Glutamate Receptors ◽  
Metabotropic Glutamate Receptors ◽  
Group Iii ◽  
Metabotropic Glutamate ◽  
Calmodulin Binding ◽  
Group I ◽  
Group Ii ◽  
Receptor Interacting Proteins ◽  
G Protein Coupled ◽  
Two Hybrid

mGluRs (metabotropic glutamate receptors) are G-protein-coupled receptors that modulate synaptic transmission. The eight mammalian mGluRs form three groups based on sequence and functional similarities: group I (1 and 5), group II (2 and 3) and group III (4, 6–8) mGluRs. In the present study, we used a Y2H (yeast two hybrid) screen to identify proteins that interact with the C-terminal intracellular tail of mGluR3. Prominent among the candidate receptor interacting proteins was calmodulin, a Ca2+ sensor known to bind identifiable sequences in group I and III mGluRs. The Y2H method was used to investigate calmodulin binding to mGluRs but failed to confirm the documented interaction with group III mGluRs. Furthermore, subsequent biochemical analysis showed that calmodulin does not interact with group II mGluRs. This illustrates that certain Ca2+-dependent interactions are not recapitulated in yeast. Moreover, it highlights the necessity for supporting biochemical data to substantiate interactions identified with Y2H methods.

scholarly journals The Nuclear Migration Protein NUDF/LIS1 Forms a Complex with NUDC and BNFA at Spindle Pole Bodies

2008 ◽  
Vol 7 (6) ◽  
pp. 1041-1052 ◽  
Author(s):  
Kerstin Helmstaedt ◽  
Karen Laubinger ◽  
Katja Voßkuhl ◽  
Özgür Bayram ◽  
Silke Busch ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Affinity Purification ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Bimolecular Fluorescence Complementation ◽  
Yeast Two Hybrid ◽  
Spindle Pole Bodies ◽  
Green Fluorescent ◽  
Two Hybrid

ABSTRACTNuclear migration depends on microtubules, the dynein motor complex, and regulatory components like LIS1 and NUDC. We sought to identify new binding partners of the fungal LIS1 homolog NUDF to clarify its function in dynein regulation. We therefore analyzed the association between NUDF and NUDC inAspergillus nidulans. NUDF and NUDC directly interacted in yeast two-hybrid experiments via NUDF's WD40 domain. NUDC-green fluorescent protein (NUDC-GFP) was localized to immobile dots in the cytoplasm and at the hyphal cortex, some of which were spindle pole bodies (SPBs). We showed by bimolecular fluorescence complementation microscopy that NUDC directly interacted with NUDF at SPBs at different stages of the cell cycle. Applying tandem affinity purification, we isolated the NUDF-associated protein BNFA (forbinding toNUDF). BNFA was dispensable for growth and for nuclear migration. GFP-BNFA fusions localized to SPBs at different stages of the cell cycle. This localization depended on NUDF, since the loss of NUDF resulted in the cytoplasmic accumulation of BNFA. BNFA did not bind to NUDC in a yeast two-hybrid assay. These results show that the conserved NUDF and NUDC proteins play a concerted role at SPBs at different stages of the cell cycle and that NUDF recruits additional proteins specifically to the dynein complex at SPBs.

FHL2 Interacts with Iaspp and Impacts the Biological Functions of Leukemia Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5111-5111
Author(s):  
Wenting Lu ◽  
Tengteng Yu ◽  
Shuang Liu ◽  
Saisai Li ◽  
Shouyun Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Transcriptional Activity ◽  
Luciferase Assay ◽  
Biological Functions ◽  
Yeast Two Hybrid ◽  
Binding Partner ◽  
Two Hybrid

Abstract Introduction iASPP played an important role in leukemogenesis in our previous study. In order to clarify its mechanism, a yeast two-hybrid screen was performed to find the binding partner of iASPP. In this study, we reported FHL2 as a novel binding partner of iASPP. The biological functions of the communication between FHL2 and iASPP were detected, and its possible mechanisms were investigated in human leukemia cell lines. Methods A yeast two-hybrid screen was performed to identify FHL2 as a novel binding partner of iASPP. Immunofluorescence, Co-IP and Western blot analysis were used to confirm the communication between FHL2 and iASPP. After FHL2 or iASPP was knocked down in K562 and Kasumi-1 cells by lentiviral, MTT assay and flow cytometry were performed to detect the proliferation, cell cycle distribution and apoptosis rate of leukemic cells, meanwhile Western blot analysis was used to analyze the level of cell cycle- and apoptotic-related proteins. Dual luciferase assay was conducted to investigate the transcriptional activity of p53 on Bax when iASPP and FHL2 were overexpressed or FHL2 was knocked down. Results FHL2 was highly expressed in K562 and Kasumi-1 cells. FHL2 and iASPP interacted with each other and co-localized in both nucleus and cytoplasm. Either FHL2 or iASPP silenced could reduce cell proliferation, induce cell cycle arrest at G0/G1 phases, and increase cell apoptosis. Western blot analysis showed that the level of p21 increased and anti-apoptotic protein Bcl-2 was reduced. Interestingly, when FHL2 was knocked down, the protein expression level of iASPP also decreased. Similarly, the expression of FHL2 would reduce when iASPP was silenced. Dual luciferase assay suggested that iASPP could reduce the transcriptional activity of p53 on Bax, furthermore, when FHL2 was knocked down at the same time, the transcriptional activity of p53 was rescued. Conclusions The interaction between FHL2 and iASPP in AML was observed for the first time. Cell proliferation reducing, cell cycle arresting at G0/G1 phases, and cell apoptosis increasing occurred in either FHL2 knockdown or iASPP knockdown. Moreover, iASPP and FHL2 participated in the regulation of the transcriptional activation function of p53. These results indicated that FHL2 might be a novel potential target for AML treatment. Disclosures No relevant conflicts of interest to declare.

Gankyrin, the 26 S proteasome, the cell cycle and cancer

2006 ◽  
Vol 34 (5) ◽  
pp. 746-748 ◽  
Author(s):  
R.J. Mayer ◽  
J. Fujita
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Cell Cycle Control ◽  
Yeast Two Hybrid ◽  
Ubiquitin Protein Ligase ◽  
Aaa Atpase ◽  
Molecular Features ◽  
Double Minute ◽  
Murine Double Minute ◽  
Two Hybrid

The known molecular players in cell-cycle control are much studied, not only to learn more about this intricate system, but also to understand the molecular features of oncogenic transformation. Infrequently, new players are discovered that change the interpretation of cell-cycle control. Gankyrin is one such player and was discovered in yeast two-hybrid screens as a new proteasomal subunit that interacts specifically with the S6b (rpt3) AAA (ATPase associated with various cellular activities) ATPase, which, with five other AAAs, are present in the so-called base of the 19 S regulator of the 26 S proteasome. Gankyrin is also the first liver oncogene. Gankyrin is found in other complexes that contain Rb (retinoblastoma protein) and the ubiquitin protein ligase Mdm2 (murine double minute 2). Gankyrin increases the hyperphosphorylation of Rb and therefore activates E2F-dependent transcription of DNA synthesis genes. Additionally, gankyrin, by binding to Mdm2, increases the ubiquitylation and degradation of p53 and prevents apoptosis. Gankyrin controls the functions of two major tumour suppressors and, when overexpressed, causes hepatocellular carcinoma.

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