scholarly journals Phosphorylation of Tyr-950 in the proteasome scaffolding protein RPN2 modulates its interaction with the ubiquitin receptor RPN13

2019 ◽  
Vol 294 (25) ◽  
pp. 9659-9665 ◽  
Author(s):  
Casey W. Hemmis ◽  
Stephanie C. Heard ◽  
Christopher P. Hill
Keyword(s):  
Anticancer Agents ◽  
Jurkat Cells ◽  
26S Proteasome ◽  
Scaffolding Protein ◽  
Binding Assays ◽  
Protein Substrates ◽  
Stress Response Pathway ◽  
Ubiquitin Receptors ◽  
Positively Charged ◽  
Specific Inhibitors

Protein substrates are targeted to the 26S proteasome through several ubiquitin receptors. One of these receptors, RPN13, is recruited to the proteasome by binding of its N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain to C-terminal residues of the scaffolding protein RPN2. The RPN13 PRU domain is followed by a flexible linker and a C-terminal deubiquitylase adaptor (DEUBAD) domain, which recruits and activates the deubiquitylase UCH37. Both RPN13 and UCH37 have been implicated in human cancers, and inhibitors of the RPN2–RPN13 interaction are being developed as potential therapeutic anticancer agents. Our current study builds on the recognition that a residue central to the RPN2–RPN13 interaction, RPN2 Tyr-950, is phosphorylated in Jurkat cells. We found that the Tyr-950 phosphorylation enhances binding to RPN13. The crystal structure of the RPN2–RPN13 pTyr-950–ubiquitin complex was determined at 1.76-Å resolution and reveals specific interactions with positively charged side chains in RPN13 that explain how phosphorylation increases binding affinity without inducing conformational change. Mutagenesis and quantitative binding assays were then used to validate the crystallographic interface. Our findings support a model in which RPN13 recruitment to the proteasome is enhanced by phosphorylation of RPN2 Tyr-950, have important implications for efforts to develop specific inhibitors of the RPN2–RPN13 interaction, and suggest the existence of a previously unknown stress-response pathway.

scholarly journals The Cdc48 unfoldase prepares well-folded protein substrates for degradation by the 26S proteasome

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Michal M. Olszewski ◽  
Cameron Williams ◽  
Ken C. Dong ◽  
Andreas Martin
Keyword(s):  
26S Proteasome ◽  
Protein Substrates

scholarly journals Protein kinase CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) induces apoptosis and caspase-dependent degradation of haematopoietic lineage cell-specific protein 1 (HS1) in Jurkat cells

2002 ◽  
Vol 364 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Maria RUZZENE ◽  
Daniele PENZO ◽  
Lorenzo A. PINNA
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Specific Inhibitor ◽  
Specific Protein ◽  
Jurkat Cells ◽  
Similar Data ◽  
Caspase Cleavage ◽  
Protein Substrates ◽  
Kinase Ck2

Incubation of Jurkat cells with 4,5,6,7-tetrabromobenzotriazole (TBB), a specific inhibitor of protein kinase CK2, induces dose-and time-dependent apoptosis as judged by several criteria. TBB-promoted apoptosis is preceded by inhibition of Ser/Thr phosphorylation of haematopoietic lineage cell-specific protein 1 (HS1) and is accompanied by caspase-dependent fragmentation of the same protein. Both effects are also observable if apoptosis is promoted by anti-Fas antibodies and by etoposide. Moreover, in vitro experiments show that HS1, once phosphorylated by CK2, becomes refractory to cleavage by caspase-3. These findings, in conjunction with similar data in the literature concerning two other CK2 protein substrates, Bid and Max, suggest that CK2 may play a general anti-apoptotic role through the generation of phosphorylated sites conferring resistance to caspase cleavage.

scholarly journals DLK induces developmental neuronal degeneration via selective regulation of proapoptotic JNK activity

2011 ◽  
Vol 194 (5) ◽  
pp. 751-764 ◽  
Author(s):  
Arundhati Sengupta Ghosh ◽  
Bei Wang ◽  
Christine D. Pozniak ◽  
Mark Chen ◽  
Ryan J. Watts ◽  
...  
Keyword(s):  
Leucine Zipper ◽  
Neuronal Degeneration ◽  
Scaffolding Protein ◽  
Axon Degeneration ◽  
Jnk Signaling ◽  
Signaling Complex ◽  
Neuronal Populations ◽  
Neuronal Homeostasis ◽  
Stress Response Pathway

The c-Jun N-terminal kinase (JNK) signaling pathway is essential for neuronal degeneration in multiple contexts but also regulates neuronal homeostasis. It remains unclear how neurons are able to dissociate proapoptotic JNK signaling from physiological JNK activity. In this paper, we show that the mixed lineage kinase dual leucine zipper kinase (DLK) selectively regulates the JNK-based stress response pathway to mediate axon degeneration and neuronal apoptosis without influencing other aspects of JNK signaling. This specificity is dependent on interaction of DLK with the scaffolding protein JIP3 to form a specialized JNK signaling complex. Local activation of DLK-based signaling in the axon results in phosphorylation of c-Jun and apoptosis after redistribution of JNK to the cell body. In contrast, regulation of axon degeneration by DLK is c-Jun independent and mediated by distinct JNK substrates. DLK-null mice displayed reduced apoptosis in multiple neuronal populations during development, demonstrating that prodegenerative DLK signaling is required in vivo.

scholarly journals Structures of the substrate-engaged 26S proteasome reveal the mechanisms for ATP hydrolysis-driven translocation

2018 ◽  
Author(s):  
Andres H. de la Peña ◽  
Ellen A. Goodall ◽  
Stephanie N. Gates ◽  
Gabriel C. Lander ◽  
Andreas Martin
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
26S Proteasome ◽  
Atp Binding ◽  
Conformational States ◽  
Phosphate Release ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Hexameric Atpase ◽  
Central Pore

AbstractThe 26S proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric ATPase motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core, while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-EM structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination, and how ATP-binding, hydrolysis, and phosphate-release events are coordinated within the AAA+ motor to induce conformational changes and propel the substrate through the central pore.

scholarly journals Antiproliferative Activities of Methanolic Extract and Fractions of Tetrapleura Tetraptera Fruit

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Anastasia Rosebud Aikins ◽  
Peggy Afua Birikorang ◽  
Mary Chama ◽  
Eunice Dotse ◽  
Abigail Anning ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Methanolic Extract ◽  
Colorimetric Assay ◽  
Jurkat Cells ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Cancer Chemotherapeutics ◽  
Tetrapleura Tetraptera ◽  
Antiproliferative Activities ◽  
Mcf 7

Most of the current cancer chemotherapeutics are associated with harsh and undesirable side effects, including toxicity and chemoresistance, driving the need for safer and more effective alternatives. In this study, the antiproliferative activities of the methanolic extract of Tetrapleura tetraptera fruits and nine different fractions (C1–C9) from the column chromatographic separation of the extract against leukemia (Jurkat) and human breast cancer (MCF-7) cell lines were investigated using a tetrazolium-based colorimetric assay. Phytochemical screening of the extract and fractions found alkaloids, carbohydrates, flavonoids, glycosides, phenols, saponins, steroids, tannins, and terpenoids in the methanolic extract. Most of the fractions exhibited antiproliferative activity (>100 μg/mL) with the Jurkat cells being more susceptible than the MCF-7 cells. Four of the collected fractions C4, C3, C5, and C2 had good selective indices in decreasing order of activity, in the case of Jurkat cells. Liquid chromatography-mass spectrometry analysis of all samples (except for C4 and C9) revealed that C1, C2, C3, and C5 each had a single component. More importantly, fractions C2, C3, and C5, which were selective to Jurkat cells, also had the same retention time of 1.846 min. Fractions C6 and C8 had two components, with C7 having four components. This study serves as a basis for further work to isolate and characterize potential anticancer agents from the fractions of extracts of T. tetraptera fruits.

scholarly journals The APC11 RING-H2 Finger Mediates E2-Dependent Ubiquitination

2000 ◽  
Vol 11 (7) ◽  
pp. 2315-2325 ◽  
Author(s):  
Joel D. Leverson ◽  
Claudio A.P. Joazeiro ◽  
Andrew M. Page ◽  
Han-kuei Huang ◽  
Philip Hieter ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
26S Proteasome ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Protein Substrates ◽  
Ubiquitin Ligase Activity ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Conjugating Enzymes

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that theSaccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.

Neutral and positively charged thiols synergize the effect of the immunomodulator AS101 as a growth inhibitor of Jurkat cells, by increasing its uptake

2007 ◽  
Vol 74 (5) ◽  
pp. 712-722 ◽  
Author(s):  
Gabi M. Frei ◽  
Ilana Lebenthal ◽  
Michael Albeck ◽  
Amnon Albeck ◽  
Benjamin Sredni
Keyword(s):  
Growth Inhibitor ◽  
Jurkat Cells ◽  
Positively Charged

scholarly journals Interaction of the Doa4 Deubiquitinating Enzyme with the Yeast 26S Proteasome

1999 ◽  
Vol 10 (3) ◽  
pp. 741-756 ◽  
Author(s):  
Feroz R. Papa ◽  
Alexander Y. Amerik ◽  
Mark Hochstrasser
Keyword(s):  
Catalytic Domain ◽  
Genetic Interaction ◽  
26S Proteasome ◽  
Deubiquitinating Enzyme ◽  
Modular Architecture ◽  
Protein Substrates ◽  
Ubiquitin Proteasome ◽  
Sizeable Fraction ◽  
Proteasome Pathway ◽  
New Yeast

The Saccharomyces cerevisiae Doa4 deubiquitinating enzyme is required for the rapid degradation of protein substrates of the ubiquitin–proteasome pathway. Previous work suggested that Doa4 functions late in the pathway, possibly by deubiquitinating (poly)-ubiquitin-substrate intermediates associated with the 26S proteasome. We now provide evidence for physical and functional interaction between Doa4 and the proteasome. Genetic interaction is indicated by the mutual enhancement of defects associated with a deletion of DOA4 or a proteasome mutation when the two mutations are combined. Physical association of Doa4 and the proteasome was investigated with a new yeast 26S proteasome purification procedure, by which we find that a sizeable fraction of Doa4 copurifies with the protease. Another yeast deubiquitinating enzyme, Ubp5, which is related in sequence to Doa4 but cannot substitute for it even when overproduced, does not associate with the proteasome. DOA4-UBP5 chimeras were made by a novel PCR/yeast recombination method and used to identify an N-terminal 310-residue domain of Doa4 that, when appended to the catalytic domain of Ubp5, conferred Doa4 function, consistent with Ubp enzymes having a modular architecture. Unlike Ubp5, a functional Doa4-Ubp5 chimera associates with the proteasome, suggesting that proteasome binding is important for Doa4 function. Together, these data support a model in which Doa4 promotes proteolysis through removal of ubiquitin from proteolytic intermediates on the proteasome before or after initiation of substrate breakdown.

A diversity of SERCA Ca2+ pump inhibitors

2011 ◽  
Vol 39 (3) ◽  
pp. 789-797 ◽  
Author(s):  
Francesco Michelangeli ◽  
J. Malcolm East
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Endocrine Disruption ◽  
Future Development ◽  
Anticancer Agents ◽  
Environmental Pollutants ◽  
Mechanisms Of Action ◽  
Vast Array ◽  
Wide Range ◽  
Specific Inhibitors

The SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) is probably the most extensively studied membrane protein transporter. There is a vast array of diverse inhibitors for the Ca2+ pump, and many have proved significant in helping to elucidate both the mechanism of transport and gaining conformational structures. Some SERCA inhibitors such as thapsigargin have been used extensively as pharmacological tools to probe the roles of Ca2+ stores in Ca2+ signalling processes. Furthermore, some inhibitors have been implicated in the cause of diseases associated with endocrine disruption by environmental pollutants, whereas others are being developed as potential anticancer agents. The present review therefore aims to highlight some of the wide range of chemically diverse inhibitors that are known, their mechanisms of action and their binding location on the Ca2+ ATPase. Additionally, some ideas for the future development of more useful isoform-specific inhibitors and anticancer drugs are presented.

scholarly journals Clastosome: A Subtype of Nuclear Body Enriched in 19S and 20S Proteasomes, Ubiquitin, and Protein Substrates of Proteasome

2002 ◽  
Vol 13 (8) ◽  
pp. 2771-2782 ◽  
Author(s):  
Miguel Lafarga ◽  
Maria Teresa Berciano ◽  
Emma Pena ◽  
Isabel Mayo ◽  
Jose G. Castaño ◽  
...  
Keyword(s):  
Proteasome Inhibitors ◽  
Cell Types ◽  
Nuclear Body ◽  
Nuclear Bodies ◽  
26S Proteasome ◽  
High Concentration ◽  
Protein Substrates ◽  
Adenovirus E1a ◽  
Ubiquitin Proteasome ◽  
Nuclear Structures

Nuclear bodies represent a heterogeneous class of nuclear structures. Herein, we describe that a subset of nuclear bodies is highly enriched in components of the ubiquitin–proteasome pathway of proteolysis. We coined the term clastosome (from the Greekklastos, broken and soma, body) to refer to this type of nuclear body. Clastosomes contain a high concentration of 1) ubiquitin conjugates, 2) the proteolytically active 20S core and the 19S regulatory complexes of the 26S proteasome, and 3) protein substrates of the proteasome. Although detected in a variety of cell types, clastosomes are scarce under normal conditions; however, they become more abundant when proteasomal activity is stimulated. In contrast, clastosomes disappear when cells are treated with proteasome inhibitors. Protein substrates of the proteasome that are found concentrated in clastosomes include the short-lived transcription factors c-Fos and c-Jun, adenovirus E1A proteins, and the PML protein. We propose that clastosomes are sites where proteolysis of a variety of protein substrates is taking place.

Sign in / Sign up

Export Citation Format

Share Document