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 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.

scholarly journals The von Hippel–Lindau Cullin-RING E3 ubiquitin ligase regulates APOBEC3 cytidine deaminases

2021 ◽  
Author(s):  
Gaël K. Scholtès ◽  
Aubrey M. Sawyer ◽  
Cristina C. Vaca ◽  
Isabelle Clerc ◽  
Meejeon Roh ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Cytidine Deaminases ◽  
Von Hippel Lindau ◽  
Ring E3

scholarly journals Axotrophin/MARCH7 acts as an E3 ubiquitin ligase and ubiquitinates tau protein in vitro impairing microtubule binding

2014 ◽  
Vol 1842 (9) ◽  
pp. 1527-1538 ◽  
Author(s):  
Katharina Flach ◽  
Ellen Ramminger ◽  
Isabel Hilbrich ◽  
Annika Arsalan-Werner ◽  
Franziska Albrecht ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Tau Protein ◽  
E3 Ubiquitin Ligase ◽  
Microtubule Binding

scholarly journals Site-specific ubiquitination exposes a linear motif to promote interferon-α receptor endocytosis

2007 ◽  
Vol 179 (5) ◽  
pp. 935-950 ◽  
Author(s):  
K.G. Suresh Kumar ◽  
Hervé Barriere ◽  
Christopher J. Carbone ◽  
Jianghuai Liu ◽  
Gayathri Swaminathan ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Interferon Α ◽  
Type I ◽  
Lysosomal Degradation ◽  
Linear Motif ◽  
Site Specific ◽  
Receptor Endocytosis ◽  
Β Receptor

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCFβTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.

Abstract 18: Synoviolin, an E3 Ubiquitin Ligase, Modulates Cardiac Myocyte Size and Restores Heart Function in Hypertrophic Cardiomyopathy

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Shirin Doroudgar ◽  
Mirko Völkers ◽  
Donna J Thuerauf ◽  
Ashley Bumbar ◽  
Mohsin Khan ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Misfolding ◽  
Cardiac Myocyte ◽  
Protein Quality ◽  
Heart Function ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
Calcium Handling ◽  
Loss Of Function

The endoplasmic reticulum (ER) is essential for protein homeostasis, or proteostasis, which governs the balance of the proteome. In addition to secreted and membrane proteins, proteins bound for many other cellular locations are also made on ER-bound ribosomes, emphasizing the importance of protein quality and quantity control in the ER. Unlike cytosolic E3 ubiquitin ligases studied in the heart, synoviolin/Hrd1, which has not been studied in the heart, is an ER transmembrane E3 ubiquitin ligase, which we found to be upregulated upon protein misfolding in cardiac myocytes. Given the strategic location of synoviolin in the ER membrane, we addressed the hypothesis that synoviolin is critical for regulating the balance of the proteome, and accordingly, myocyte size. We showed that in vitro, adenovirus-mediated overexpression of synoviolin decreased cardiac myocyte size and protein synthesis, but unlike atrophy-related ubiquitin ligases, synoviolin did not increase global protein degradation. Furthermore, targeted gene therapy using adeno-associated virus 9 (AAV9) showed that overexpression of synoviolin in the left ventricle attenuated maladaptive cardiac hypertrophy and preserved cardiac function in mice subjected to trans-aortic constriction (AAV9-control TAC = 22.5 ± 6.2% decrease in EF vs. AAV9-synoviolin TAC at 6 weeks post TAC; P<0.001), and decreased mTOR activity. Since calcium is a major regulator of cardiac myocyte size, we examined the effects of synoviolin gain- or loss-of-function, using AAV9-synoviolin, or an miRNA designed to knock down synoviolin, respectively. While synoviolin gain-of-function did not affect calcium handling in isolated adult myocytes, synoviolin loss-of-function increased calcium transient amplitude (P<0.01), prolonged spark duration (P<0.001), and increased spark width (P<0.001). Spark frequency and amplitude were unaltered upon synoviolin gain- or loss-of-function. Whereas SR calcium load was unaltered by synoviolin loss-of-function, SERCA-mediated calcium removal was reduced (P<0.05). In conclusion, our studies suggest that in the heart, synoviolin is 1) a critical component of proteostasis, 2) a novel determinant of cardiac myocyte size, and 3) necessary for proper calcium handling.

CBL mutations drive PI3K/AKT signaling via increased interaction with LYN and PIK3R1

Blood ◽  
2021 ◽  
Author(s):  
Roger Belizaire ◽  
Sebastian Hassan John Koochaki ◽  
Namrata D. Udeshi ◽  
Alexis Vedder ◽  
Lei Sun ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ubiquitin Ligase ◽  
Therapeutic Potential ◽  
Mutant Cell ◽  
E3 Ubiquitin Ligase ◽  
Akt Signaling ◽  
Allelic Series ◽  
Genetic Ablation

CBL encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and non-receptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10-20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, PIK3R1 recruitment, and downstream PI3K/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine-kinase binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

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