scholarly journals Structure-Guided Design and Optimization of Small Molecules Targeting the Protein–Protein Interaction between the von Hippel–Lindau (VHL) E3 Ubiquitin Ligase and the Hypoxia Inducible Factor (HIF) Alpha Subunit with in Vitro Nanomolar Affinities

2014 ◽  
Vol 57 (20) ◽  
pp. 8657-8663 ◽  
Author(s):  
Carles Galdeano ◽  
Morgan S. Gadd ◽  
Pedro Soares ◽  
Salvatore Scaffidi ◽  
Inge Van Molle ◽  
...  
Keyword(s):  
Small Molecules ◽  
Ubiquitin Ligase ◽  
Protein Interaction ◽  
Hypoxia Inducible Factor ◽  
E3 Ubiquitin Ligase ◽  
Alpha Subunit ◽  
Von Hippel Lindau ◽  
Design And Optimization ◽  
Protein Protein Interaction

scholarly journals Methods for addressing the protein-protein interaction between histone deacetylase 6 and ubiquitin

2017 ◽  
Author(s):  
Carolina dos S. Passos ◽  
Nathalie Deschamps ◽  
Yun Choi ◽  
Robert E. Cohen ◽  
Remo Perozzo ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Stress Response ◽  
Small Molecules ◽  
Histone Deacetylase ◽  
Protein Interaction ◽  
Potential Role ◽  
Histone Deacetylase 6 ◽  
Protein Protein Interaction ◽  
Binding Function

AbstractHistone deacetylase 6 (HDAC6) is a cytoplasmic HDAC isoform able to remove acetyl groups from cellular substrates such as α-tubulin. In addition to the two deacetylase domains, HDAC6 has a C-terminal zinc-finger ubiquitin (Ub)-binding domain (ZnF-UBP) able to recognize free Ub. HDAC6-Ub interaction is thought to function in regulating the elimination of misfolded proteins during stress response through the aggresome pathway. Small molecules inhibiting deacetylation by HDAC6 were shown to reduce aggresomes, but the interplay between HDAC6 catalytic activity and Ub-binding function is not fully understood. Here we describe two methods to measure the HDAC6-Ub interaction in vitro using full-length HDAC6. Both methods were effective for screening inhibitors of the HDAC6-Ub protein-protein interaction independently of the catalytic activity. Our results suggest a potential role for the HDAC6 deacetylase domains in modulating HDAC6-Ub interaction. This new aspect of HDAC6 regulation can be targeted to address the roles of HDAC6-Ub interaction in normal and disease conditions.

scholarly journals Diverse Effects of Mutations in Exon II of the von Hippel-Lindau (VHL) Tumor Suppressor Gene on the Interaction of pVHL with the Cytosolic Chaperonin and pVHL-Dependent Ubiquitin Ligase Activity

2002 ◽  
Vol 22 (6) ◽  
pp. 1947-1960 ◽  
Author(s):  
William J. Hansen ◽  
Michael Ohh ◽  
Javid Moslehi ◽  
Keiichi Kondo ◽  
William G. Kaelin ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Ubiquitin Ligase ◽  
Hypoxia Inducible Factor ◽  
Detectable Effect ◽  
Missense Mutations ◽  
Subsequent Formation ◽  
Von Hippel Lindau ◽  
Ubiquitin Ligase Activity

ABSTRACT We examined the biogenesis of the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL) in vitro and in vivo. pVHL formed a complex with the cytosolic chaperonin containing TCP-1 (CCT or TRiC) en route to assembly with elongin B/C and the subsequent formation of the VCB-Cul2 ubiquitin ligase. Blocking the interaction of pVHL with elongin B/C resulted in accumulation of pVHL within the CCT complex. pVHL present in purified VHL-CCT complexes, when added to rabbit reticulocyte lysate, proceeded to form VCB and VCB-Cul2. Thus, CCT likely functions, at least in part, by retaining VHL chains pending the availability of elongin B/C for final folding and/or assembly. Tumor-associated mutations within exon II of the VHL syndrome had diverse effects upon the stability and/or function of pVHL-containing complexes. First, a pVHL mutant lacking the entire region encoded by exon II did not bind to CCT and yet could still assemble into complexes with elongin B/C and elongin B/C-Cul2. Second, a number of tumor-derived missense mutations in exon II did not decrease CCT binding, and most had no detectable effect upon VCB-Cul2 assembly. Many exon II mutants, however, were found to be defective in the binding to and subsequent ubiquitination of hypoxia-inducible factor 1α (HIF-1α), a substrate of the VCB-Cul2 ubiquitin ligase. We conclude that the selection pressure to mutate VHL exon II during tumorigenesis does not relate to loss of CCT binding but may reflect quantitative or qualitative defects in HIF binding and/or in pVHL-dependent ubiquitin ligase activity.

scholarly journals Playing Tag with HIF: The VHL Story

2002 ◽  
Vol 2 (3) ◽  
pp. 131-135 ◽  
Author(s):  
Sherri K. Leung ◽  
Michael Ohh
Keyword(s):  
Ubiquitin Ligase ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
E3 Ubiquitin Ligase ◽  
Suppressor Gene ◽  
Tumour Suppressor Gene ◽  
Matrix Assembly ◽  
Von Hippel Lindau ◽  
Ubiquitin Ligase Complex

Inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL is the cause of inherited VHL disease and is associated with sporadic kidney cancer. pVHL is found in a multiprotein complex with elongins B/C, Cul2, and Rbx1 forming an E3 ubiquitin ligase complex called VEC. This modular enzyme targets theαsubunits of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction. Consequently, tumour cells lacking functional pVHL overproduce the products of HIF-target genes such as vascular endothelial growth factor (VEGF), which promotes angiogenesis. This likely accounts for the hypervascular nature of VHL-associated neoplasms. Although pVHL has been linked to the cell-cycle, differentiation, and the regulation of extracellular matrix assembly, microenvironment pH, and tissue invasiveness, this review will focus on the recent insights into the molecular mechanisms governing the E3 ubiquitin ligase function of VEC.

Erythroid-specific 5-aminolevulinate synthase protein is stabilized by low oxygen and proteasomal inhibition

2005 ◽  
Vol 83 (5) ◽  
pp. 620-630 ◽  
Author(s):  
Mohamed Abu-Farha ◽  
Jacques Niles ◽  
William G Willmore
Keyword(s):  
Ubiquitin Ligase ◽  
Specific Activity ◽  
E3 Ubiquitin Ligase ◽  
K562 Cells ◽  
Turnover Time ◽  
Low Oxygen ◽  
Flag Epitope ◽  
Aminolevulinate Synthase ◽  
Von Hippel Lindau

5-aminolevulinate synthase (ALAS; E.C. 2.3.1.37) catalyzes the first and rate-limiting step of heme synthesis within the mitochondria. Two isozymes of ALAS, encoded by separate genes, exist. ALAS1 is ubiquitously expressed and provides heme for cytochromes and other hemoproteins. ALAS2 is expressed exclusively in erythroid cells and synthesizes heme specifically for haemoglobin. A database search for proteins potentially regulated by oxygen tension revealed that ALAS2 contained a sequence of amino acids (LXXLAP where L is leucine, X is any amino acid, A is alanine, and P is proline) not occurring in ALAS1, which may be hydroxylated under normoxic conditions (21% O2) and target the enzyme for ubiquitination and degradation by the proteasome. We examined protein turnover of ALAS2 in the presence of cycloheximide in K562 cells. Normoxic ALAS2 had a turnover time of approximately 36 h. Hypoxia (1% O2) and inhibition of the proteasome increased both the stability and the specific activity of ALAS2 (greater than 2- and 7-fold, respectively, over 72 h of treatment). Mutation of a key proline within the LXXLAP sequence of ALAS2 also stabilized the protein beyond 36 h under normoxic conditions. The von Hippel-Lindau (vHL) protein was immunoprecipitated with FLAG epitope-tagged ALAS2 produced in normoxic cells but not in hypoxic cells, suggesting that the ALAS2 is hydroxylated under normoxic conditions and targeted for ubiquitination by the E3 ubiquitin ligase system. ALAS2 could also be ubiquitinated under normoxia using an in vitro ubiquitination assay. The present study provides evidence that ALAS2 is broken down under normoxic conditions by the proteasome and that the prolyl-4-hydroxylase/vHL E3 ubiquitin ligase pathway may be involved.Key words: erythroid-specific 5-aminolevulinate synthase, hypoxia, hydroxylation, prolyl-4-hydroxylases, E3 ubiquitin ligases, von Hippel-Lindau protein, proteasome.

scholarly journals Coordinate Regulation of the Oxygen-Dependent Degradation Domains of Hypoxia-Inducible Factor 1α

2005 ◽  
Vol 25 (15) ◽  
pp. 6415-6426 ◽  
Author(s):  
Denise A. Chan ◽  
Patrick D. Sutphin ◽  
Shing-Erh Yen ◽  
Amato J. Giaccia
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Turnover ◽  
Hypoxia Inducible Factor ◽  
E3 Ubiquitin Ligase ◽  
Domain Swapping ◽  
Coordinate Regulation ◽  
Von Hippel Lindau ◽  
Primary Means ◽  
Oxygen Tensions ◽  
Different Levels

ABSTRACT Oxygen-dependent proteolysis is the primary means of regulating the hypoxia-inducible factor (HIF) family of transcription factors. The alpha-subunit of HIF factor 1 (HIF-1) contains two highly conserved oxygen-dependent degradation domains (402 ODD and 564 ODD), each of which includes a proline that is hydroxylated in the presence of oxygen, allowing the von Hippel-Lindau (VHL) E3 ubiquitin ligase to interact and target HIF-1α to the proteasome for degradation. Mutation of either proline is sufficient to partially stabilize HIF-1α under conditions of normoxia, but the specific contributions of each hydroxylation event to the regulation of HIF-1α are unknown. Here we show that the two ODDs of HIF-1α have independent yet interactive roles in the regulation of HIF-1α protein turnover, with the relative involvement of each ODD depending on the levels of oxygen. Using hydroxylation-specific antibodies, we found that under conditions of normoxia proline 564 is hydroxylated prior to proline 402, and mutation of proline 564 results in a significant reduction in the hydroxylation of proline 402. Mutation of proline 402, however, has little effect on the hydroxylation of proline 564. To determine whether the more rapid hydroxylation of the proline 564 under conditions of normoxia is due to a preference for the particular sequence surrounding proline 564 or for that site within the protein, we exchanged the degradation domains within the full-length HIF-1α protein. In these domain-swapping experiments, prolyl hydroxylase domain 1 (PHD1) and PHD2 preferentially hydroxylated the proline located in the site of the original 564 ODD, while PHD3 preferred the proline 564 sequence, regardless of its location. At limiting oxygen tensions, we found that proline 402 exhibits an oxygen-dependent decrease in hydroxylation at higher oxygen tensions relative to proline 564 hydroxylation. These results indicate that hydroxylation of proline 402 is highly responsive to physiologic changes in oxygen and, therefore, plays a more important role in HIF-1α regulation under conditions of hypoxia than under conditions of normoxia. Together, these findings demonstrate that each hydroxylated proline of HIF-1α has a distinct activity in controlling HIF-1α stability in response to different levels of oxygenation.

scholarly journals Exploring the Ubiquitin-Proteasome System (UPS) through PROTAC Technology

2020 ◽  
Vol 74 (4) ◽  
pp. 274-277
Author(s):  
Carlotta Cecchini ◽  
Sébastien Tardy ◽  
Valentina Ceserani ◽  
Jean-Philippe Theurillat ◽  
Leonardo Scapozza
Keyword(s):  
Renal Cell Carcinoma ◽  
Small Molecules ◽  
Ubiquitin Ligase ◽  
Active Sites ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Proteasome System ◽  
Classical Approach ◽  
Hypoxia Inducible Factors ◽  
Von Hippel Lindau ◽  
Ubiquitin Proteasome

In the context of dysregulated ubiquitylation, the accumulation of oncogenic substrates can lead to tumorigenesis. In particular, mutations in Von Hippel-Lindau (VHL) E3 ubiquitin ligase are related to overexpression of hypoxia-inducible factors (HIF-1α and HIF-2α) which is evolving into renal cell carcinoma (RCC). The classical approach of drug discovery focuses on the development of highly selective small molecules able to bind and to inhibit enzymatic active sites. This strategy faces limitations in the context of ' undruggable ' proteins, which are challenging to target. The discovery of Proteolysis Targeting Chimeras (PROTACs) as an alternative strategy to induce selective protein degradation is presented as a working hypothesis to understand further the UbiquitinProteasome System (UPS) and eventually counteract RCC cancer lacking VHL ubiquitin ligase.

scholarly journals Hypoxia-Associated Factor, a Novel E3-Ubiquitin Ligase, Binds and Ubiquitinates Hypoxia-Inducible Factor 1α, Leading to Its Oxygen-Independent Degradation

2008 ◽  
Vol 28 (23) ◽  
pp. 7081-7095 ◽  
Author(s):  
Mei Yee Koh ◽  
Bryant G. Darnay ◽  
Garth Powis
Keyword(s):  
Ubiquitin Ligase ◽  
Cellular Response ◽  
Hypoxia Inducible Factor ◽  
E3 Ubiquitin Ligase ◽  
Degradation Pathway ◽  
Proliferating Cells ◽  
Associated Factor ◽  
Hypoxia Inducible Factor 1Α

ABSTRACT The hypoxia-inducible factor 1α (HIF-1α) is the master regulator of the cellular response to hypoxia. A key regulator of HIF-1α is von Hippel-Lindau protein (pVHL), which mediates the oxygen-dependent, proteasomal degradation of HIF-1α in normoxia. Here, we describe a new regulator of HIF-1α, the hypoxia-associated factor (HAF), a novel E3-ubiquitin ligase that binds HIF-1α leading to its proteasome-dependent degradation irrespective of cellular oxygen tension. HAF, a protein expressed in proliferating cells, binds and ubiquitinates HIF-1α in vitro, and both binding and E3 ligase activity are mediated by HAF amino acids 654 to 800. Furthermore, HAF overexpression decreases HIF-1α levels in normoxia and hypoxia in both pVHL-competent and -deficient cells, whereas HAF knockdown increases HIF-1α levels in normoxia, hypoxia, and under epidermal growth factor stimulation. In contrast, HIF-2α is not regulated by HAF. In vivo, tumor xenografts from cells overexpressing HAF show decreased levels of HIF-1α accompanied by decreased tumor growth and angiogenesis. Therefore, HAF is the key mediator of a new HIF-1α-specific degradation pathway that degrades HIF-1α through a new, oxygen-independent mechanism.

scholarly journals Targeting the von Hippel–Lindau E3 Ubiquitin Ligase Using Small Molecules To Disrupt the VHL/HIF-1α Interaction

2012 ◽  
Vol 134 (10) ◽  
pp. 4465-4468 ◽  
Author(s):  
Dennis L. Buckley ◽  
Inge Van Molle ◽  
Peter C. Gareiss ◽  
Hyun Seop Tae ◽  
Julien Michel ◽  
...  
Keyword(s):  
Small Molecules ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Von Hippel Lindau

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

scholarly journals Therapeutically actionable signaling node to rescue AURKA driven loss of primary cilia in VHL-deficient cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pratim Chowdhury ◽  
Dimuthu Perera ◽  
Reid T. Powell ◽  
Tia Talley ◽  
Durga Nand Tripathi ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Primary Cilia ◽  
Mtor Inhibitor ◽  
E3 Ubiquitin Ligase ◽  
Xenograft Model ◽  
Aurora Kinase ◽  
Tumor Burden ◽  
Aurora Kinase A ◽  
Von Hippel Lindau ◽  
In Cells

AbstractLoss of primary cilia in cells deficient for the tumor suppressor von Hippel Lindau (VHL) arise from elevated Aurora Kinase A (AURKA) levels. VHL in its role as an E3 ubiquitin ligase targets AURKA for degradation and in the absence of VHL, high levels of AURKA result in destabilization of the primary cilium. We identified NVP-BEZ235, a dual PI3K/AKT and mTOR inhibitor, in an image-based high throughput screen, as a small molecule that restored primary cilia in VHL-deficient cells. We identified the ability of AKT to modulate AURKA expression at the transcript and protein level. Independent modulation of AKT and mTOR signaling decreased AURKA expression in cells confirming AURKA as a new signaling node downstream of the PI3K cascade. Corroborating these data, a genetic knockdown of AKT in cells deficient for VHL rescued the ability of these cells to ciliate. Finally, inhibition of AKT/mTOR using NVP-BEZ235 was efficacious in reducing tumor burden in a 786-0 xenograft model of renal cell carcinoma. These data highlight a previously unappreciated signaling node downstream of the AKT/mTOR pathway via AURKA that can be targeted in VHL-null cells to restore ciliogenesis.

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