scholarly journals Drosophila Nedd4-long reduces Amphiphysin levels in muscles and leads to impaired T-tubule formation

2016 ◽  
Vol 27 (6) ◽  
pp. 907-918 ◽  
Author(s):  
Frozan Safi ◽  
Alina Shteiman-Kotler ◽  
Yunan Zhong ◽  
Konstantin G. Iliadi ◽  
Gabrielle L. Boulianne ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Tubule Formation ◽  
Splice Isoforms ◽  
Unique Region ◽  
Postsynaptic Protein ◽  
Binding Partners ◽  
N Terminus ◽  
Direct Binding ◽  
T Tubules ◽  
Neuromuscular Synaptogenesis

Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4-short (dNedd4S) and -long (dNedd4Lo). DNedd4Lo has a unique N-terminus containing a Pro-rich region. We previously showed that whereas dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, we searched for binding partners to the N-terminal unique region of dNedd4Lo in larval lysates using mass spectrometry and identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains and regulates muscle transverse tubule (T-tubule) formation in flies. We validated the interaction by coimmunoprecipitation and showed direct binding between dAmph-SH3 domain and dNedd4Lo N-terminus. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and impaired T-tubule formation, phenocopying amph-null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C→A). We propose that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function.

scholarly journals Regulation of SH3PX1 by dNedd4-long at the Drosophila neuromuscular junction

2018 ◽  
Vol 294 (5) ◽  
pp. 1739-1752 ◽  
Author(s):  
Samantha S. Wasserman ◽  
Alina Shteiman-Kotler ◽  
Kathryn Harris ◽  
Konstantin G. Iliadi ◽  
Avinash Persaud ◽  
...  
Keyword(s):  
Neurotransmitter Release ◽  
Neuromuscular Junctions ◽  
Cell Periphery ◽  
Middle Region ◽  
Binding Partners ◽  
Direct Binding ◽  
Specific Regulation ◽  
Neuromuscular Synaptogenesis

Drosophila Nedd4 (dNedd4) is a HECT E3 ubiquitin ligase present in two major isoforms: short (dNedd4S) and long (dNedd4Lo), with the latter containing two unique regions (N terminus and Middle). Although dNedd4S promotes neuromuscular synaptogenesis (NMS), dNedd4Lo inhibits it and impairs larval locomotion. To explain how dNedd4Lo inhibits NMS, MS analysis was performed to find its binding partners and identified SH3PX1, which binds dNedd4Lo unique Middle region. SH3PX1 contains SH3, PX, and BAR domains and is present at neuromuscular junctions, where it regulates active zone ultrastructure and presynaptic neurotransmitter release. Here, we demonstrate direct binding of SH3PX1 to the dNedd4Lo Middle region (which contains a Pro-rich sequence) in vitro and in cells, via the SH3PX1-SH3 domain. In Drosophila S2 cells, dNedd4Lo overexpression reduces SH3PX1 levels at the cell periphery. In vivo overexpression of dNedd4Lo post-synaptically, but not pre-synaptically, reduces SH3PX1 levels at the subsynaptic reticulum and impairs neurotransmitter release. Unexpectedly, larvae that overexpress dNedd4Lo post-synaptically and are heterozygous for a null mutation in SH3PX1 display increased neurotransmission compared with dNedd4Lo or SH3PX1 mutant larvae alone, suggesting a compensatory effect from the remaining SH3PX1 allele. These results suggest a post-synaptic–specific regulation of SH3PX1 by dNedd4Lo.

scholarly journals Protein Binding Partners of Dysregulated miRNAs in Parkinson’s Disease Serum

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 791
Author(s):  
Wolfgang P. Ruf ◽  
Axel Freischmidt ◽  
Veselin Grozdanov ◽  
Valerie Roth ◽  
Sarah J. Brockmann ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Protein Binding ◽  
Neurodegenerative Diseases ◽  
Extracellular Vesicles ◽  
Abundance Pattern ◽  
Binding Partners ◽  
Direct Binding ◽  
Unsupervised Approach ◽  
High Degree

Accumulating evidence suggests that microRNAs (miRNAs) are a contributing factor to neurodegenerative diseases. Although altered miRNA profiles in serum or plasma have been reported for several neurodegenerative diseases, little is known about the interaction between dysregulated miRNAs and their protein binding partners. We found significant alterations of the miRNA abundance pattern in serum and in isolated serum-derived extracellular vesicles of Parkinson’s disease (PD) patients. The differential expression of miRNA in PD patients was more robust in serum than in isolated extracellular vesicles and could separate PD patients from healthy controls in an unsupervised approach to a high degree. We identified a novel protein interaction partner for the strongly dysregulated hsa-mir-4745-5p. Our study provides further evidence for the involvement of miRNAs and HNF4a in PD. The demonstration that miRNA-protein binding might mediate the pathologic effects of HNF4a both by direct binding to it and by binding to proteins regulated by it suggests a complex role for miRNAs in pathology beyond the dysregulation of transcription.

Abstract P495:

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Magda C. Diaz ◽  
Raúl Flores-Vergara ◽  
Ivonne Olmedo ◽  
Zully Pedrozo
Keyword(s):  
Heart Failure ◽  
Cardiac Function ◽  
Protein Content ◽  
Cardiac Tissue ◽  
Negative Regulator ◽  
Mrna Levels ◽  
Tubule Formation ◽  
T Tubules ◽  
Factor C ◽  
Old Mice

Background: Transverse tubules (T-tubules) play a key role in cardiac contractility. The expression of Bridging integrator 1 (BIN1), specifically, the cardiac isoforms BIN1+13 and BIN1+ 13+17 promote the formation and ultrastructure of T-tubules and it has been described that the transcriptional factor c-Myc may be a negative regulator of BIN1 expression. Polycystin-1 (PC1) is a mechanosensor in cardiomyocytes, with a crucial role to maintain cardiac function. Our aim was to determine whether PC1 regulates BIN1-induced T-tubule formation by a c-Myc-regulated mechanism. Methods: We used adult C57BL/6 cardiomyocyte-specific knockout mice (PC1 KO). Cardiac function was tested by echocardiography, whereas protein and mRNA content of BIN1, PC1 and c-Myc were measured using western blot and qRT-PCR, respectively. T-tubules were analyzed by transmission electron microscopy (TEM). For statistical analyses, t-test or one-way ANOVA followed by Tukey's test were used. Differences were considered significant at p < 0.05. Results: Survival of PC1 KO mice decreased dramatically after 7 months of age, with clear symptoms of dilated cardiomyopathy and heart failure (decreased fractional shortening and ejection fraction). Ventricular cardiac tissue of PC1 KO mice without (< 7-month-old mice) and with symptoms (7-9-month-old mice) of heart failure (HF) was associated with reduced levels of BIN1 protein content. Total BIN1 mRNA in PC1 KO mice without symptoms did not show differences as compared to controls, but it was significantly decreased in mice with symptoms of HF. Moreover, PC1 KO mice with and without symptoms showed decreased BIN1+13 mRNA levels, whereas BIN1+13+17 mRNA was only increased in mice without symptoms. These changes in BIN1 isoforms were related with T-tubules, which showed increased lumen and decreased intra-luminal density in PC1 KO mice with and without symptoms. Furthermore, c-Myc protein content was increased in cardiac tissue of PC1 KO mice. Conclusion: PC1 may be a regulator of the differential expression of cardiac isoforms of BIN1 in cardiomyocytes by a mechanism that involves negative regulation of c-Myc, related to loss of T-tubule ultrastructure and heart failure development.

scholarly journals Direct In Vitro Binding of Full-Length Human Immunodeficiency Virus Type 1 Nef Protein to CD4 Cytoplasmic Domain

2001 ◽  
Vol 75 (8) ◽  
pp. 3960-3964 ◽  
Author(s):  
Andrea Preusser ◽  
Lars Briese ◽  
Andreas S. Baur ◽  
Dieter Willbold
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Human Immunodeficiency Virus ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Immunodeficiency Virus ◽  
N Terminus ◽  
Human Immunodeficiency Viruses ◽  
Direct Binding

ABSTRACT The Nef protein of the simian and human immunodeficiency viruses is known to directly bind and downregulate the CD4 receptor. Although the molecular mechanism is well understood, direct binding of Nef and CD4 is difficult to demonstrate and is believed to be of low affinity. Applying nuclear magnetic resonance and fluorescence spectroscopy, we biophysically reevaluated the CD4-Nef complex and found the dissociation constant to be in the submicromolar range. We conclude that additional, so far disregarded residues in the N terminus of Nef are important for interaction with CD4.

scholarly journals Mapping of a spectrin-binding domain of human erythrocyte membrane protein 4.2

2002 ◽  
Vol 364 (3) ◽  
pp. 841-847 ◽  
Author(s):  
Debabrata MANDAL ◽  
Prasun K. MOITRA ◽  
Joyoti BASU
Keyword(s):  
Erythrocyte Membrane ◽  
Synthetic Peptide ◽  
Membrane Skeleton ◽  
Band 3 Protein ◽  
Fusion Peptides ◽  
Human Erythrocyte Membrane ◽  
Glutathione S Transferase ◽  
Protein 4.2 ◽  
N Terminus ◽  
Direct Binding

Protein 4.2 is a major component of the red blood cell membrane skeleton. Deficiency of protein 4.2 is linked with a variety of hereditary haemolytic anaemias. However, the interactions of protein 4.2 with other proteins of the erythrocyte membrane remain poorly understood. The major membrane-binding site for protein 4.2 resides on the cytoplasmic domain of band 3. Protein 4.2 interacts directly with spectrin in solution, suggesting that it stabilizes interactions between the membrane skeleton and the erythrocyte membrane. A 30kDa polypeptide, with its N-terminus corresponding to amino acid residue 269, derived by partial proteolysis of protein 4.2, was found to interact with biotinylated spectrin in gel renaturation assays. A series of overlapping glutathione S-transferase fusion peptides were constructed, and an α-helical domain encompassing residues 470–492 was found to be instrumental in mediating protein 4.2—spectrin interactions. Direct binding of a synthetic peptide, with the sequence corresponding to residues 470–492, to spectrin and the ability of the peptide to inhibit spectrin binding of protein 4.2 confirmed that these residues are crucial in mediating protein 4.2—spectrin interactions.

scholarly journals NF-κB activation is a turn on for vaccinia virus phosphoprotein A49 to turn off NF-κB activation

2019 ◽  
Vol 116 (12) ◽  
pp. 5699-5704 ◽  
Author(s):  
Sarah Neidel ◽  
Hongwei Ren ◽  
Alice A. Torres ◽  
Geoffrey L. Smith
Keyword(s):  
Vaccinia Virus ◽  
Ubiquitin Ligase ◽  
Molecular Mimicry ◽  
E3 Ubiquitin Ligase ◽  
Virus Protein ◽  
Biological Regulation ◽  
Turn On ◽  
N Terminus ◽  
Iκbα Phosphorylation ◽  
Necessary And Sufficient

Vaccinia virus protein A49 inhibits NF-κB activation by molecular mimicry and has a motif near the N terminus that is conserved in IκBα, β-catenin, HIV Vpu, and some other proteins. This motif contains two serines, and for IκBα and β-catenin, phosphorylation of these serines enables recognition by the E3 ubiquitin ligase β-TrCP. Binding of IκBα and β-catenin by β-TrCP causes their ubiquitylation and thereafter proteasome-mediated degradation. In contrast, HIV Vpu and VACV A49 are not degraded. This paper shows that A49 is phosphorylated at serine 7 but not serine 12 and that this is necessary and sufficient for binding β-TrCP and antagonism of NF-κB. Phosphorylation of A49 S7 occurs when NF-κB signaling is activated by addition of IL-1β or overexpression of TRAF6 or IKKβ, the kinase needed for IκBα phosphorylation. Thus, A49 shows beautiful biological regulation, for it becomes an NF-κB antagonist upon activation of NF-κB signaling. The virulence of viruses expressing mutant A49 proteins or lacking A49 (vΔA49) was tested. vΔA49 was attenuated compared with WT, but viruses expressing A49 that cannot bind β-TrCP or bind β-TrCP constitutively had intermediate virulence. So A49 promotes virulence by inhibiting NF-κB activation and by another mechanism independent of S7 phosphorylation and NF-κB antagonism. Last, a virus lacking A49 was more immunogenic than the WT virus.

scholarly journals Espin Contains an Additional Actin-binding Site in Its N Terminus and Is a Major Actin-bundling Protein of the Sertoli Cell–Spermatid Ectoplasmic Specialization Junctional Plaque

1999 ◽  
Vol 10 (12) ◽  
pp. 4327-4339 ◽  
Author(s):  
Bin Chen ◽  
Anli Li ◽  
Dennis Wang ◽  
Min Wang ◽  
Lili Zheng ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Sertoli Cell ◽  
Actin Binding ◽  
Actin Stress Fiber ◽  
Splice Isoforms ◽  
Actin Bundles ◽  
N Terminus ◽  
Ectoplasmic Specialization ◽  
Actin Binding Site

The espins are actin-binding and -bundling proteins localized to parallel actin bundles. The 837-amino-acid “espin” of Sertoli cell–spermatid junctions (ectoplasmic specializations) and the 253-amino-acid “small espin” of brush border microvilli are splice isoforms that share a C-terminal 116-amino-acid actin-bundling module but contain different N termini. To investigate the roles of espin and its extended N terminus, we examined the actin-binding and -bundling properties of espin constructs and the stoichiometry and developmental accumulation of espin within the ectoplasmic specialization. An espin construct bound to F-actin with an approximately threefold higher affinity (K d = ∼70 nM) than small espin and was ∼2.5 times more efficient at forming bundles. The increased affinity appeared to be due to an additional actin-binding site in the N terminus of espin. This additional actin-binding site bound to F-actin with a K d of ∼1 μM, decorated actin stress fiber-like structures in transfected cells, and was mapped to a peptide between the two proline-rich peptides in the N terminus of espin. Espin was detected at ∼4–5 × 106 copies per ectoplasmic specialization, or ∼1 espin per 20 actin monomers and accumulated there coincident with the formation of parallel actin bundles during spermiogenesis. These results suggest that espin is a major actin-bundling protein of the Sertoli cell–spermatid ectoplasmic specialization.

scholarly journals Mms1 and Mms22 stabilize the replisome during replication stress

2011 ◽  
Vol 22 (13) ◽  
pp. 2396-2408 ◽  
Author(s):  
Jessica A. Vaisica ◽  
Anastasija Baryshnikova ◽  
Michael Costanzo ◽  
Charles Boone ◽  
Grant W. Brown
Keyword(s):  
Dna Replication ◽  
Ubiquitin Ligase ◽  
Replication Fork ◽  
E3 Ubiquitin Ligase ◽  
Replication Stress ◽  
Replication Forks ◽  
Replication Fork Progression ◽  
Binding Partners ◽  
Efficient Recovery ◽  
Stalled Replication Forks

Mms1 and Mms22 form a Cul4Ddb1-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101Mms1/Mms22ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1—Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.

scholarly journals Senescence-inducing stress promotes proteolysis of phosphoglycerate mutase via ubiquitin ligase Mdm2

2014 ◽  
Vol 204 (5) ◽  
pp. 729-745 ◽  
Author(s):  
Takumi Mikawa ◽  
Takeshi Maruyama ◽  
Koji Okamoto ◽  
Hitoshi Nakagama ◽  
Matilde E. Lleonart ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Warburg Effect ◽  
Cultured Cells ◽  
Glycolytic Enzyme ◽  
Phosphoglycerate Mutase ◽  
Proliferative Potential ◽  
Downstream Effector ◽  
Direct Binding ◽  
The Warburg Effect

Despite the well-documented clinical significance of the Warburg effect, it remains unclear how the aggressive glycolytic rates of tumor cells might contribute to other hallmarks of cancer, such as bypass of senescence. Here, we report that, during oncogene- or DNA damage–induced senescence, Pak1-mediated phosphorylation of phosphoglycerate mutase (PGAM) predisposes the glycolytic enzyme to ubiquitin-mediated degradation. We identify Mdm2 as a direct binding partner and ubiquitin ligase for PGAM in cultured cells and in vitro. Mutations in PGAM and Mdm2 that abrogate ubiquitination of PGAM restored the proliferative potential of primary cells under stress conditions and promoted neoplastic transformation. We propose that Mdm2, a downstream effector of p53, attenuates the Warburg effect via ubiquitination and degradation of PGAM.

scholarly journals Checkpoint Protein BubR1 Acts Synergistically with Mad2 to Inhibit Anaphase-promoting Complex

2002 ◽  
Vol 13 (3) ◽  
pp. 755-766 ◽  
Author(s):  
Guowei Fang
Keyword(s):  
Ubiquitin Ligase ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Metaphase Plate ◽  
Anaphase Promoting Complex ◽  
Checkpoint Control ◽  
Sister Chromatids ◽  
Direct Binding ◽  
And Function

The spindle assembly checkpoint monitors the attachment of kinetochores to the mitotic spindle and the tension exerted on kinetochores by microtubules and delays the onset of anaphase until all the chromosomes are aligned at the metaphase plate. The target of the checkpoint control is the anaphase-promoting complex (APC)/cyclosome, a ubiquitin ligase whose activation by Cdc20 is required for separation of sister chromatids. In response to activation of the checkpoint, Mad2 binds to and inhibits Cdc20-APC. I show herein that in checkpoint-arrested cells, human Cdc20 forms two separate, inactive complexes, a lower affinity complex with Mad2 and a higher affinity complex with BubR1. Purified BubR1 binds to recombinant Cdc20 and this interaction is direct. Binding of BubR1 to Cdc20 inhibits activation of APC and this inhibition is independent of its kinase activity. Quantitative analysis indicates that BubR1 is 12-fold more potent than Mad2 as an inhibitor of Cdc20. Although at high protein concentrations BubR1 and Mad2 each is sufficient to inhibit Cdc20, BubR1 and Mad2 mutually promote each other's binding to Cdc20 and function synergistically at physiological concentrations to quantitatively inhibit Cdc20-APC. Thus, BubR1 and Mad2 act cooperatively to prevent premature separation of sister chromatids by directly inhibiting APC.

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