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. 2016 Dec;1866(2):208-220.
doi: 10.1016/j.bbcan.2016.09.004. Epub 2016 Sep 20.

New Insights Into Protein Hydroxylation and Its Important Role in Human Diseases

Free PMC article

New Insights Into Protein Hydroxylation and Its Important Role in Human Diseases

Giada Zurlo et al. Biochim Biophys Acta. .
Free PMC article


Protein hydroxylation is a post-translational modification catalyzed by 2-oxoglutarate-dependent dioxygenases. The hydroxylation modification can take place on various amino acids, including but not limited to proline, lysine, asparagine, aspartate and histidine. A classical example of this modification is hypoxia inducible factor alpha (HIF-α) prolyl hydroxylation, which affects HIF-α protein stability via the Von-Hippel Lindau (VHL) tumor suppressor pathway, a Cullin 2-based E3 ligase adaptor protein frequently mutated in kidney cancer. In addition to protein stability regulation, protein hydroxylation may influence other post-translational modifications or the kinase activity of the modified protein (such as Akt and DYRK1A/B). In other cases, protein hydroxylation may alter protein-protein interaction and its downstream signaling events in vivo (such as OTUB1, MAPK6 and eEF2K). In this review, we highlight the recently identified protein hydroxylation targets and their pathophysiological roles, especially in cancer settings. Better understanding of protein hydroxylation will help identify novel therapeutic targets and their regulation mechanisms to foster development of more effective treatment strategies for various human cancers.

Keywords: Human Cancer; Hydroxylation; Hypoxia Inducible Factor alpha; Von-Hippel Lindau (VHL).

Conflict of interest statement

The authors declare that they have no conflict of interest.


Figure 1
Figure 1. HIF-1α regulation by hydroxylation
(A) In the presence of oxygen, HIF-1α is hydroxylated by the prolyl hydroxylases PHD/EglN1, 2, 3 on prolines 402 and 564. This leads to HIF-1α recognition by the ubiquitin ligase complex composed of the von Hippel Lindau protein (cullin2) and RBX1 (RING-box1), which promotes the polyubiquitination of HIF-1α followed by its proteasomal degradation. (B) The lack of oxygen prevents the hydroxylation of HIF-1α by PHD, leading to its stabilization. HIF-1α can then migrate to the nucleus and associate with ARNT (aryl hydrocarbon receptor nuclear translocator) and the cofactor p300/CBP (cyclic AMP response element-binding protein). The HIF-1 complex binds to and induces the transcription of genes containing hypoxia-responsive elements (HRE) in their promoter region. (C) FIH (factor inhibiting HIF) can hydroxylate HIF on the asparagine 803 (Asn803) residue, which affects its binding with the transcriptional co-activator p300/CBP, thereby inhibiting HIF transcriptional activity.
Figure 2
Figure 2. FOXO3a regulation by hydroxylation
(A) The prolyl hydroxylase PHD1/EglN2 hydroxylates FOXO3a (Forkhead box-O3a) transcription factor on two proline residues (Pro426 and 437), thus preventing the binding with USP9x (ubiquitin specific peptidase 9, X-linked) deubiquitinase. This promotes FOXO3a ubiquitination and proteasomal degradation. (B) In the case of low activity levels of EglN2 (PHD1), e. g. under hypoxia, USP9x binds to FOXO3a thus preventing its ubiquitination and increasing its stability. FOXO3a being a transcriptional repressor for cyclin D1, its stabilization leads to a decreased cyclin D1 expression.
Figure 3
Figure 3. DYRK1A/B regulation by hydroxylation
(A) PHD1/EglN2 triggers DYRK1A and B (dual-specificity tyrosine-(Y)-phosphorylation regulated kinases 1A and B) hydroxylation, thus promoting their phosphorylation and kinase activity. Subsequently, ID2 (inhibitor of differentiation 2) is phosphorylated and prevented from interacting with pVHL ubiquitinase complex, therefore preserving pVHL-dependent HIF-2α ubiquitination and proteasomal degradation. (B) Under hypoxia or PHD1 depletion, DYRK1A and B cannot be efficiently hydroxylated or phosphorylated, thus preventing ID2 phosphorylation on Thr27. This promotes ID2 binding with pVHL that disturbs the scaffold protein CUL2 interaction with pVHL E3 ligase complex. As a consequence, pVHL cannot efficiently ubiquitinate HIF-2α. HIF-2α accumulation is then followed by transcriptional regulation of its downstream target gene expression.
Figure 4
Figure 4. NDRG3 regulation by hydroxylation
(A) NDRG3 (N-Myc downstream-regulated gene) can be hydroxylated on proline 294, potentially by PHD2/EglN1. The hydroxylated NDRG3 binds to pVHL, which promotes NDRG3 ubiquitination and degradation. (B) Hypoxia can lead to the accumulation of lactate that directly binds NDRG3, thus preventing its interaction with the pVHL complex. Therefore, NDRG3 is stabilized and may contribute to downstream RAF-ERK1/2 kinase signaling.
Figure 5
Figure 5. Akt regulation by hydroxylation
(A) Akt can be hydroxylated on Pro125 and Pro313 residues by PHD2/EglN1. pVHL and the phosphatase PP2A interact with hydroxylated Akt and inhibit Akt activity, as demonstrated by the decrease of Akt phosphorylation on threonine 308 (Thr308). (B) The lack of oxygen or functional pVHL prevents Akt hydroxylation and binding with pVHL and PP2A, therefore leading to the increase of Akt Thr308 phosphorylation and activity and contributing to increased cell proliferation and tumorigenesis.
Figure 6
Figure 6. EPOR, MAPK6 and RIPK4 regulation by hydroxylation
(A) PHD3/EglN3 -mediated prolyl hydroxylation on EPOR (erythropoietin receptor) Pro419 and Pro426 residues facilitates EPOR binding with pVHL complex, hereby causing its proteasomal degradation. PHD3/EglN3 depletion stabilizes EPOR and, therefore, stimulates JAK/STAT signaling pathway-driven erythropoiesis. (B) MAPK6 (Mitogen-activated protein kinase 6) can be hydroxylated by PHD3/EglN3 on proline 25 (Pro25), which potentially leads to its dissociation from HUWE1 (HECT, UBA and WWE domain containing 1) E3 ubiquitin protein ligase, thus protecting it from proteasomal degradation. (C) RIPK4 hydroxylation by FIH affects RIPK4 activity, therefore contributing to Wnt signaling pathway activity modulation.
Figure 7
Figure 7. eEF2K, β and γ-actin regulation by hydroxylation
(A) Under normoxic condition, eEF2K (eukaryotic elongation factor 2 kinase) can be hydroxylated on proline 98 (Pro98), which disrupts its binding with calmodulin and inhibits its kinase activity. Therefore, eEF2 cannot be efficiently phosphorylated and protein synthesis proceeds. Hypoxic condition prevents eEF2K hydroxylation, allowing eEF2K interaction with calmodulin. Activated eEF2K phosphorylates eEF2 on threonine 56 (Thr56) therefore inhibiting its translation elongation activity. (B) β and γ-actin isoforms are hydroxylated by PHD3/EglN3 on Pro307 and Pro322 residues, which reduces actin polymerization. PHD3/EglN3 depletion leads to increased F-actin expression and cell motility in HeLa cells.

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