Progesterone Induces Porcine Sperm Release from Oviduct Glycans in a Proteasome-Dependent Manner

Reproduction ◽  
2021 ◽  
Author(s):  
Momal Sharif ◽  
Karl Kerns ◽  
Peter Sutovsky ◽  
Nicolai Bovin ◽  
David J. Miller
Keyword(s):  
Proteasome Inhibitors ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Dependent Manner ◽  
Sperm Release ◽  
Ubiquitin Proteasome ◽  
Glycan Receptors ◽  
Branched Structure ◽  
Oviduct Cell

In mammals, the oviduct retains sperm forming a reservoir from which they are released in synchrony with ovulation. However, the mechanisms underlying sperm release are unclear. Herein, we first examined in greater detail the release of sperm from the oviduct reservoir by sex steroids, and second, if the ubiquitin-proteasome system (UPS) mediates this release in vitro. Sperm were allowed to bind to oviductal cells or immobilized oviduct glycans, either bi-SiaLN or a suLeX, and then challenged with steroids in the presence or absence of proteasome inhibitors. Previously, we have demonstrated that progesterone-induced sperm release from oviduct cells and immobilized glycans in a steroid-specific manner. Herein we found that release of sperm from an immobilized oviduct glycan, a 6-sialylated branched structure, and from immobilized fibronectin was inhibited by the CatSper blocker NNC 055-0396, akin to the previously reported ability of NNC 055-0396 to inhibit sperm release from another oviduct glycan, sulfated Lewis X trisaccharide. Thus, CatSper may be required for release from a variety of adhesion systems. One possible mechanism for sperm release is that glycan receptors on sperm are degraded by proteasomes or shed from the sperm surface by proteasomal degradation. Accordingly, the inhibition of proteasomal degradation blocked sperm release from oviduct cell aggregates and both immobilized oviduct glycans as well as fibronectin. In summary, progesterone-induced sperm release required both active CatSper channels and proteasomal degradation, suggesting that hyperactivation and proteolysis are vital parts of the mechanism by which sperm move from the oviduct reservoir to the site of fertilization.

scholarly journals The AAA-ATPase p97 is essential for outer mitochondrial membrane protein turnover

2011 ◽  
Vol 22 (3) ◽  
pp. 291-300 ◽  
Author(s):  
Shan Xu ◽  
Guihong Peng ◽  
Yang Wang ◽  
Shengyun Fang ◽  
Mariusz Karbowski
Keyword(s):  
Mitochondrial Membrane ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Degradation Pathway ◽  
Outer Mitochondrial Membrane ◽  
Aaa Atpase ◽  
Associated Proteins ◽  
Ubiquitin Proteasome ◽  
Molecular Components

Recent studies have revealed a role for the ubiquitin/proteasome system in the regulation and turnover of outer mitochondrial membrane (OMM)-associated proteins. Although several molecular components required for this process have been identified, the mechanism of proteasome-dependent degradation of OMM-associated proteins is currently unclear. We show that an AAA-ATPase, p97, is required for the proteasomal degradation of Mcl1 and Mfn1, two unrelated OMM proteins with short half-lives. A number of biochemical assays, as well as imaging of changes in localization of photoactivable GFP-fused Mcl1, revealed that p97 regulates the retrotranslocation of Mcl1 from mitochondria to the cytosol, prior to, or concurrent with, proteasomal degradation. Mcl1 retrotranslocation from the OMM depends on the activity of the ATPase domain of p97. Furthermore, p97-mediated retrotranslocation of Mcl1 can be recapitulated in vitro, confirming a direct mitochondrial role for p97. Our results establish p97 as a novel and essential component of the OMM-associated protein degradation pathway.

scholarly journals β-TrCP-mediated ubiquitination and degradation of Dlg5 regulates hepatocellular carcinoma cell proliferation

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Dongping Wang ◽  
Qi Zhang ◽  
Fenfen Li ◽  
Chan Wang ◽  
Changming Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Xenograft Model ◽  
Ubiquitin Proteasome System ◽  
Tumor Xenograft ◽  
Dependent Manner ◽  
Ubiquitin Proteasome ◽  
Novel Strategy ◽  
Hcc Cells

Abstract Background Discs large homolog 5 (Dlg5) is a member of the membrane-associated guanylate kinase (MAGUK) adaptor family of proteins and its deregulation has been implicated in the malignancy of several cancer types. Dlg5 was down-regulated in hepatocellular carcinoma (HCC) and lower Dlg5 expression was associated with poor survival of HCC patients. However, how to regulate Dlg5 remains largely unknown. Methods The co-immunoprecipitation assay was used to determine the interaction between Dlg5 and β-TrCP. The in vivo ubiquitination assay was performed to determine the regulation of Dlg5 by β-TrCP. CCK-8 and colony formation assay were implemented to detect the biological effect of Dlg5 on the growth of HCC cells in vitro. The effect of Dlg5 on HCC tumor growth in vivo was studied in a tumor xenograft model in mice. Results Here we report that Dlg5 is regulated by the ubiquitin proteasome system and depletion of either Cullin 1 or β-TrCP led to increased levels of Dlg5. β-TrCP regulated Dlg5 protein stability by targeting it for ubiquitination and subsequent destruction in a phosphorylation-dependent manner. We further demonstrated a crucial role of Ser730 in the non-canonical phosphodegron of Dlg5 in governing β-TrCP-mediated Dlg5 degradation. Importantly, failure to degrade Dlg5 significantly inhibited HCC cells proliferation both in vitro and in vivo. Conclusion Collectively, our finding provides a novel molecular mechanism for the negative regulation of Dlg5 by β-TRCP in HCC cells. It further suggests that preventing Dlg5 degradation could be a possible novel strategy for clinical treatment of HCC.

scholarly journals Effect of Proteasome Inhibitors With Different Chemical Structures on the Ubiquitin–Proteasome System In Vitro

2010 ◽  
Vol 47 (2) ◽  
pp. 358-367 ◽  
Author(s):  
V. Csizmadia ◽  
E. Csizmadia ◽  
L. Silverman ◽  
C. Simpson ◽  
A. Raczynski ◽  
...  
Keyword(s):  
Proteasome Inhibitors ◽  
Ubiquitin Proteasome System ◽  
Chemical Structures ◽  
Ubiquitin Proteasome

scholarly journals Ubiquitination of both Adeno-Associated Virus Type 2 and 5 Capsid Proteins Affects the Transduction Efficiency of Recombinant Vectors

2002 ◽  
Vol 76 (5) ◽  
pp. 2043-2053 ◽  
Author(s):  
Ziying Yan ◽  
Roman Zak ◽  
G. W. Gant Luxton ◽  
Teresa C. Ritchie ◽  
Ursula Bantel-Schaal ◽  
...  
Keyword(s):  
Proteasome Inhibitors ◽  
Ubiquitin Proteasome System ◽  
Capsid Proteins ◽  
Transduction Efficiency ◽  
Viral Capsid ◽  
Adeno Associated Virus ◽  
Ubiquitin Proteasome ◽  
Viral Capsid Proteins

ABSTRACT In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.

scholarly journals An F-Box Protein, Mdm30, Interacts with TREX Subunit Sub2 To Regulate Cellular Abundance Cotranscriptionally in Orchestrating mRNA Export Independently of Splicing and Mitochondrial Function

2020 ◽  
Vol 40 (7) ◽  
Author(s):  
Jannatul Ferdoush ◽  
Rwik Sen ◽  
Geetha Durairaj ◽  
Priyanka Barman ◽  
Amala Kaja ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Mitochondrial Dynamics ◽  
Mrna Export ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Dependent Manner ◽  
Direct Role ◽  
Transcriptional Inhibition ◽  
Ubiquitin Proteasome ◽  
F Box Protein

ABSTRACT Although an F-box protein, Mdm30, is found to regulate ubiquitylation of the Sub2 component of TREX (transcription-export) complex for proteasomal degradation in stimulation of mRNA export, it remains unknown whether such ubiquitin-proteasome system (UPS) regulation of Sub2 occurs cotranscriptionally via its interaction with Mdm30. Further, it is unclear whether impaired UPS regulation of Sub2 in the absence of Mdm30 alters mRNA export via splicing defects of export factors and/or mitochondrial dynamics/function, since Sub2 controls mRNA splicing and Mdm30 regulates mitochondrial aggregation. Here, we show that Mdm30 interacts with Sub2, and temporary shutdown of Mdm30 enhances Sub2’s abundance and impairs mRNA export. Likewise, Sub2’s abundance is increased following transcriptional inhibition. These results support Mdm30’s direct role in regulation of Sub2’s cellular abundance in a transcription-dependent manner. Consistently, the chromatin-bound Sub2 level is increased in the absence of Mdm30. Further, we find that Mdm30 does not facilitate splicing of export factors. Moreover, Mdm30 does not have a dramatic effect on mitochondrial respiration/function, and mRNA export occurs in the absence of Fzo1, which is required for mitochondrial dynamics/respiration. Collective results reveal that Mdm30 interacts with Sub2 for proteasomal degradation in a transcription-dependent manner to promote mRNA export independently of splicing or mitochondrial function, thus advancing our understanding of mRNA export.

scholarly journals Breakdown of Filamentous Myofibrils by the UPS–Step by Step

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 110
Author(s):  
Dina Aweida ◽  
Shenhav Cohen
Keyword(s):  
Protein Quality ◽  
Protein Structures ◽  
Ubiquitin Proteasome System ◽  
Surface Proteins ◽  
Catalytic Core ◽  
Complex Protein ◽  
Ubiquitin Proteasome ◽  
Aaa Atpases

Protein degradation maintains cellular integrity by regulating virtually all biological processes, whereas impaired proteolysis perturbs protein quality control, and often leads to human disease. Two major proteolytic systems are responsible for protein breakdown in all cells: autophagy, which facilitates the loss of organelles, protein aggregates, and cell surface proteins; and the ubiquitin-proteasome system (UPS), which promotes degradation of mainly soluble proteins. Recent findings indicate that more complex protein structures, such as filamentous assemblies, which are not accessible to the catalytic core of the proteasome in vitro, can be efficiently degraded by this proteolytic machinery in systemic catabolic states in vivo. Mechanisms that loosen the filamentous structure seem to be activated first, hence increasing the accessibility of protein constituents to the UPS. In this review, we will discuss the mechanisms underlying the disassembly and loss of the intricate insoluble filamentous myofibrils, which are responsible for muscle contraction, and whose degradation by the UPS causes weakness and disability in aging and disease. Several lines of evidence indicate that myofibril breakdown occurs in a strictly ordered and controlled manner, and the function of AAA-ATPases is crucial for their disassembly and loss.

scholarly journals A Yeast-Based Functional Assay to Study Plant N-Degron – N-Recognin Interactions

2022 ◽  
Vol 12 ◽  
Author(s):  
Aida Kozlic ◽  
Nikola Winter ◽  
Theresia Telser ◽  
Jakob Reimann ◽  
Katrin Rose ◽  
...  
Keyword(s):  
Ubiquitin Proteasome System ◽  
Functional Assay ◽  
The Novel ◽  
In Planta ◽  
Amino Terminal ◽  
Weak Binding ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Proteasome ◽  
The Stability

The N-degron pathway is a branch of the ubiquitin-proteasome system where amino-terminal residues serve as degradation signals. In a synthetic biology approach, we expressed ubiquitin ligase PRT6 and ubiquitin conjugating enzyme 2 (AtUBC2) from Arabidopsis thaliana in a Saccharomyces cerevisiae strain with mutation in its endogenous N-degron pathway. The two enzymes re-constitute part of the plant N-degron pathway and were probed by monitoring the stability of co-expressed GFP-linked plant proteins starting with Arginine N-degrons. The novel assay allows for straightforward analysis, whereas in vitro interaction assays often do not allow detection of the weak binding of N-degron recognizing ubiquitin ligases to their substrates, and in planta testing is usually complex and time-consuming.

scholarly journals Ccr4 is a novel shuttle factor required for ubiquitin-dependent proteolysis by the 26S proteasome

2020 ◽  
Author(s):  
Ganapathi Kandasamy ◽  
Ashis Kumar Pradhan ◽  
R Palanimurugan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Degradation ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Rna Degradation ◽  
26S Proteasome ◽  
Cellular Proteins ◽  
Cellular Homeostasis ◽  
Substrate Degradation ◽  
Ubiquitin Proteasome

AbstractDegradation of short-lived and abnormal proteins are essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Furthermore, abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4 protein is a known component of the Ccr4-Not complex, which has established roles in transcription, mRNA de-adenylation and RNA degradation etc. Excitingly in this study, we show that Ccr4 protein has a novel function as a shuttle factor that promotes ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4 in Saccharomyces cerevisiae. Additionally, we show that Ccr4 binds to cellular ubiquitin conjugates and the proteasome. In contrast to Ccr4, most other subunits of the Ccr4-Not complex proteins are dispensable for UFD substrate degradation. From our findings we conclude that Ccr4 functions in the UPS as a shuttle factor targeting ubiquitylated substrates for proteasomal degradation.

scholarly journals Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation

2005 ◽  
Vol 25 (2) ◽  
pp. 854-864 ◽  
Author(s):  
Sandrine Marchetti ◽  
Clotilde Gimond ◽  
Jean-Claude Chambard ◽  
Thomas Touboul ◽  
Danièle Roux ◽  
...  
Keyword(s):  
Map Kinases ◽  
Fluorescent Protein ◽  
Proteasomal Degradation ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Double Mutation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.

Abstract 37: Centrosome Destabilization Through the Ubiquitin-Proteasome Pathway Regulates Proplatelet Production

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Kellie R Machlus ◽  
Prakrith Vijey ◽  
Thomas Soussou ◽  
Joseph E Italiano
Keyword(s):  
Protein Degradation ◽  
Proteasome Inhibitors ◽  
Aurora Kinase ◽  
Proteasome Activity ◽  
Major Pathway ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Proplatelet Formation

Background: Proteasome inhibitors such as bortezomib, a chemotherapeutic used to treat multiple myeloma, induce thrombocytopenia within days of initiation. The mechanism for this thrombocytopenia has been tied to data revealing that proteasome activity is essential for platelet formation. The major pathway of selective protein degradation uses ubiquitin as a marker that targets proteins for proteolysis by the proteasome. This pathway is previously unexplored in megakaryocytes (MKs). Objectives: We aim to define the mechanism by which the ubiquitin-proteasome pathway affects MK maturation and platelet production. Results: Pharmacologic inhibition of proteasome activity blocks proplatelet formation in megakaryocytes. To further characterize how this degradation was occurring, we probed distinct ubiquitin pathways. Inhibition of the ubiquitin-activating enzyme E1 significantly inhibited proplatelet formation up to 73%. In addition, inhibition of the deubiquitinase proteins UCHL5 and USP14 significantly inhibited proplatelet formation up to 83%. These data suggest that an intact ubiquitin pathway is necessary for proplatelet formation. Proteomic and polysome analyses of MKs undergoing proplatelet formation revealed a subset of proteins decreased in proplatelet-producing megakaryocytes, consistent with data showing that protein degradation is necessary for proplatelet formation. Specifically, the centrosome stabilizing proteins Aurora kinase (Aurk) A/B, Tpx2, Cdk1, and Plk1 were decreased in proplatelet-producing MKs. Furthermore, inhibition of AurkA and Plk1, but not Cdk1, significantly inhibited proplatelet formation in vitro over 83%. Conclusions: We hypothesize that proplatelet formation is triggered by centrosome destabilization and disassembly, and that the ubiquitin-proteasome pathway plays a crucial role in this transformation. Specifically, regulation of the AurkA/Plk1/Tpx2 pathway may be key in centrosome integrity and initiation of proplatelet formation. Determination of the mechanism by which the ubiquitin-proteasome pathway regulates the centrosome and facilitates proplatelet formation will allow us to design better strategies to target and reverse thrombocytopenia.

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