scholarly journals FKBPL and FKBP8 regulate DLK degradation and neuronal responses to axon injury

2021 ◽  
Author(s):  
Bohm Lee ◽  
Yeonsoo Oh ◽  
Eunhye Cho ◽  
Aaron DiAntonio ◽  
Valeria Cavalli ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Axon Regeneration ◽  
Kinase Domain ◽  
Interacting Proteins ◽  
Bafilomycin A1 ◽  
Neuronal Responses ◽  
Yeast Two Hybrid ◽  
Two Hybrid ◽  
Hybrid Screening

DLK is a key regulator of axon regeneration and degeneration in response to neuronal injury. To understand the molecular mechanisms controlling the DLK function, we performed yeast two-hybrid screening analysis and identified FKBPL as a DLK-binding protein that bound to the kinase domain and inhibited the kinase enzymatic activity of DLK. FKBPL regulated DLK stability through ubiquitin-dependent DLK degradation. We tested other members in the FKBP protein family and found that FKBP8 also induced DLK degradation as FKBPL did. We found that Lysine 271 residue in the kinase domain of DLK was a major site of ubiquitination and SUMO3-conjugation and responsible for FKBP8-mediated degradation. In vivo overexpression of FKBP8 delayed progression of axon degeneration and neuronal death following axotomy in sciatic and optic nerves, respectively, although axon regeneration efficiency was not enhanced. This research identified FKBPL and FKBP8 as new DLK-interacting proteins that regulated DLK stability by MG-132 or bafilomycin A1-sensitive protein degradation.

scholarly journals BPAG1n4 is essential for retrograde axonal transport in sensory neurons

2003 ◽  
Vol 163 (2) ◽  
pp. 223-229 ◽  
Author(s):  
Jia-Jia Liu ◽  
Jianqing Ding ◽  
Anthony S. Kowal ◽  
Timothy Nardine ◽  
Elizabeth Allen ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Sensory Neurons ◽  
Axonal Degeneration ◽  
Retrograde Axonal Transport ◽  
Binding Assays ◽  
Yeast Two Hybrid ◽  
Cytoskeletal Networks ◽  
Two Hybrid ◽  
Hybrid Screening

Disruption of the BPAG1 (bullous pemphigoid antigen 1) gene results in progressive deterioration in motor function and devastating sensory neurodegeneration in the null mice. We have previously demonstrated that BPAG1n1 and BPAG1n3 play important roles in organizing cytoskeletal networks in vivo. Here, we characterize functions of a novel BPAG1 neuronal isoform, BPAG1n4. Results obtained from yeast two-hybrid screening, blot overlay binding assays, and coimmunoprecipitations demonstrate that BPAG1n4 interacts directly with dynactin p150Glued through its unique ezrin/radixin/moesin domain. Studies using double immunofluorescent microscopy and ultrastructural analysis reveal physiological colocalization of BPAG1n4 with dynactin/dynein. Disruption of the interaction between BPAG1n4 and dynactin results in severe defects in retrograde axonal transport. We conclude that BPAG1n4 plays an essential role in retrograde axonal transport in sensory neurons. These findings might advance our understanding of pathogenesis of axonal degeneration and neuronal death.

scholarly journals Interaction of Mouse Polycomb-Group (Pc-G) Proteins Enx1 and Enx2 with Eed: Indication for Separate Pc-G Complexes

1998 ◽  
Vol 18 (6) ◽  
pp. 3572-3579 ◽  
Author(s):  
Maarten van Lohuizen ◽  
Marieke Tijms ◽  
Jan Willem Voncken ◽  
Armin Schumacher ◽  
Terry Magnuson ◽  
...  
Keyword(s):  
G Proteins ◽  
Polycomb Group ◽  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Sex Combs ◽  
Embryonic Ectoderm Development ◽  
Transient Interactions ◽  
Embryonic Ectoderm ◽  
Two Hybrid

ABSTRACT The Polycomb group (Pc-G) constitutes an important, functionally conserved group of proteins, required to stably maintain inactive homeobox genes repressed during development. Drosophila extra sex combs (esc) and its mammalian homolog embryonic ectoderm development (eed) are special Pc-G members, in that they are required early during development when Pc-G repression is initiated, a process that is still poorly understood. To get insight in the molecular function of Eed, we searched for Eed-interacting proteins, using the yeast two-hybrid method. Here we describe the specific in vivo binding of Eed to Enx1 and Enx2, two mammalian homologs of the essential DrosophilaPc-G gene Enhancer-of-zeste[E(z)]. No direct biochemical interactions were found between Eed/Enx and a previously characterized mouse Pc-G protein complex, containing several mouse Pc-G proteins includingmouse polyhomeotic (Mph1). This suggests that different Pc-G complexes with distinct functions may exist. However, partial colocalization of Enx1 and Mph1 to subnuclear domains may point to more transient interactions between these complexes, in support of a bridging role for Enx1.

scholarly journals Automated Yeast Two-hybrid Screening for Nuclear Receptor-interacting Proteins

2004 ◽  
Vol 4 (2) ◽  
pp. 205-213 ◽  
Author(s):  
Michael Albers ◽  
Harald Kranz ◽  
Ingo Kober ◽  
Carmen Kaiser ◽  
Martin Klink ◽  
...  
Keyword(s):  
Nuclear Receptor ◽  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Receptor Interacting Proteins ◽  
Two Hybrid ◽  
Hybrid Screening

scholarly journals Hantavirus nucleocapsid protein interacts with the Fas-mediated apoptosis enhancer Daxx

2002 ◽  
Vol 83 (4) ◽  
pp. 759-766 ◽  
Author(s):  
Xiao-Dong Li ◽  
Tomi P. Mäkelä ◽  
Deyin Guo ◽  
Rabah Soliymani ◽  
Vesa Koistinen ◽  
...  
Keyword(s):  
Binding Sites ◽  
Nuclear Localization Signal ◽  
Nucleocapsid Protein ◽  
Molecular Mechanisms ◽  
Cellular Proteins ◽  
Yeast Two Hybrid ◽  
Localization Signal ◽  
Carboxyl Terminal ◽  
Two Hybrid ◽  
Hybrid Screening

Hantaviruses cause two severe diseases, haemorrhagic fever with renal syndrome in Eurasia and hantavirus pulmonary syndrome in the Americas. To understand more about the molecular mechanisms that lead to these diseases, the associations of Puumala virus nucleocapsid protein (PUUV-N) with cellular proteins were studied by yeast two-hybrid screening. Daxx, known as an apoptosis enhancer, was identified from a HeLa cDNA library and its interaction with PUUV-N was confirmed by GST pull-down assay, co-immunoprecipitation and co-localization studies. Furthermore, domains of interaction were mapped to the carboxyl-terminal region of 142 amino acids in Daxx and the carboxyl-terminal 57 residues in PUUV-N, respectively. In pepscan assays, the binding sites of Daxx to PUUV-N were mapped further to two lysine-rich regions, of which one overlaps the sequence of the predicted nuclear localization signal of Daxx. These data suggest a direct link between host cell machinery and a hantavirus structural component.

scholarly journals Identification of neuroglobin-interacting proteins using yeast two-hybrid screening

Neuroscience ◽  
2012 ◽  
Vol 200 ◽  
pp. 99-105 ◽  
Author(s):  
Z. Yu ◽  
N. Liu ◽  
Y. Wang ◽  
X. Li ◽  
X. Wang
Keyword(s):  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Two Hybrid ◽  
Hybrid Screening

Proteome Profiling Identified Amyloid-β Protein Precursor as a Novel Binding Partner and Modulator of VGLUT1

2021 ◽  
pp. 1-58
Author(s):  
Jin-wu Zhou ◽  
Man Zhao ◽  
Wen-liang Rang ◽  
Xiao-yan Zhang ◽  
Zhen-ming Liu ◽  
...  
Keyword(s):  
Yeast Two Hybrid ◽  
In Vivo Models ◽  
Immunoprecipitation Assay ◽  
Protein Partners ◽  
Candidate Protein ◽  
Split Ubiquitin ◽  
Two Hybrid ◽  
Hybrid Screening

Background: The toxicity of excessive glutamate release has been implicated in various acute and chronic neurodegenerative conditions. Vesicular glutamate transporters (VGLUTs) are the major mediators for the uptake of glutamate into synaptic vesicles. However, the dynamics and mechanism of this process in glutamatergic neurons are still largely unknown. Objective: This study aimed to investigate the candidate protein partners of VGLUT1 and their regulatory roles in the vesicles in rat brain. Methods: Pull down assay, co-immunoprecipitation assay, or split-ubiquitin membrane yeast two hybrid screening coupled with nanoRPLC-MS/MS were used to identify the candidate protein partners of VGLUT1 in the vesicles in rat brain. The in vitro and in vivo models were used to test effects of AβPP, Atp6ap2, Gja1, and Synataxin on VGLUT1 expression. Results: A total of 255 and 225 proteins and 172 known genes were identified in the pull down assay, co-immunoprecipitation assay, or split-ubiquitin yeast two-hybrid screening respectively. The physiological interactions of SV2A, Syntaxin 12, Gja1, AβPP, and Atp6ap2 to VGLUT1 were further confirmed. Knockdown of Atp6ap2, Gja1, and Synataxin increased VGLUT1 mRNA expression and only knockdown of AβPP increased both mRNA and protein levels of VGLUT1 in PC12 cells. The regulatory function of AβPP on VGLUT1 expression was further confirmed in the in vitro and in vivo models. Conclusion: These results elucidate that the AβPP and VGLUT1 interacts at vesicular level and AβPP plays a role in the regulation of VGLUT1 expression which is essential for maintaining vesicular activities.

scholarly journals A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteins in vivo

2005 ◽  
Vol 102 (46) ◽  
pp. 16729-16734 ◽  
Author(s):  
F. Vignols ◽  
C. Brehelin ◽  
Y. Surdin-Kerjan ◽  
D. Thomas ◽  
Y. Meyer
Keyword(s):  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Knockout Strain ◽  
Two Hybrid

scholarly journals Targeting of C-Terminal Binding Protein (CtBP) by ARF Results in p53-Independent Apoptosis

2006 ◽  
Vol 26 (6) ◽  
pp. 2360-2372 ◽  
Author(s):  
Seema Paliwal ◽  
Sandhya Pande ◽  
Ramesh C. Kovi ◽  
Norman E. Sharpless ◽  
Nabeel Bardeesy ◽  
...  
Keyword(s):  
Tumor Suppression ◽  
Negative Regulator ◽  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Induced Apoptosis ◽  
Interfering Rna ◽  
Two Hybrid ◽  
Potent Tumor

ABSTRACT ARF encodes a potent tumor suppressor that antagonizes MDM2, a negative regulator of p53. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF or p53 singly null mice, results in a broadened tumor spectrum and decreased tumor latency. To investigate the mechanism of p53-independent tumor suppression by ARF, potential interacting proteins were identified by yeast two-hybrid screen. The antiapoptotic transcriptional corepressor C-terminal binding protein 2 (CtBP2) was identified, and ARF interactions with both CtBP1 and CtBP2 were confirmed in vitro and in vivo. Interaction with ARF resulted in proteasome-dependent CtBP degradation. Both ARF-induced CtBP degradation and CtBP small interfering RNA led to p53-independent apoptosis in colon cancer cells. ARF induction of apoptosis was dependent on its ability to interact with CtBP, and reversal of ARF-induced CtBP depletion by CtBP overexpression abrogated ARF-induced apoptosis. CtBP proteins represent putative targets for p53-independent tumor suppression by ARF.

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