E3 Ubiquitin Ligase Enzyme Chip-mediated Hif-1¦Á Degradation Mechanism

Tumor oxygenation is known to be a key regulator that provides cellularmicroenvironment and mediates tumor susceptibility to invasion and metastasis.Physiologically, a great proportion of tumor cells are under hypoxia, which leads tomany functional adaptations. Moreover, hypoxic stress also induces gene activation thatis modulated by hypoxia inducible factors (HIFs). Up to date, HIFs have been dividedinto three categories: HIF 1 (4), HIF 2 (Endothelial PAS domain protein 1, EPAS1) (5)and HIF 3 (Inhibitory PAS domain protein, IPAS) (6). Active HIF is a heterodimercomposed of HIFαand HIFβ, the former is oxygen sensitive and extremely unstablein normoxia; the latter, also named ARNT (aryl hydrocarbon receptor nucleartranslocator), is constitutively expressed. HIF 1αhas five functional domains: bHLH(basic helix loop helix) and PAS (PER ARNT SIM) domains are used to bind to HIFβ,bHLH is also essential for DNA binding (7); NAD (N terminal activation domain) andCAD (C terminal activation domain) domains are used to exert their transactivationfunction; ODD (oxygen dependent degradation) domain that partially overlaps withNAD is subject to posttranslational modifications and has great effect on the stability ofHIF 1α(8).Classically, HIF 1αcan be hydroxylated at Pro402 and Pro564 in ODD domain byPHDs (prolyl hydroxylase domain containing proteins), which is necessary to bind to anE3 ubiquitin ligase, VHL (Von Hippel–Lindau) tumor suppressor protein, fordegradation in normoxia (9). Besides this, HIF 1αalso can be modified byphosphorylation, S-nitrosylation and acetylation (8). As acetylation is mentioned, ARD1(arrest defective 1 protein) was found to bind to HIF 1αand acetylate Lys532 in ODD domain that decreased its protein expression in mouse model (10). However, ARD1seems to have no impact on HIF 1αstability in human tumor cells (11, 12, 13). Morerecently, it was reported that SSAT1/2 (Spermidine/spermine N acetyltransferase 1/2)participated in HIF 1αdegradation (14, 15). Although whether SSAT1/2 can acetylateHIF 1αis still not clear, their N acetyltransferase activity was a prerequisite for thedegradation of HIF 1α. On the other hand, Histone deacetylases (HDACs) also involvein reversible acetylation of HIF 1α. HDAC1 can deacetylate HIF 1αand prevent it fromMTA1 (metastasis associated protein 1) mediated degradation (16). In addition,knockdown of HDAC4 can renew acetylation of HIF 1αand induce its degradation(17).CHIP (carboxyl terminus of Hsc70 interacting protein), also termed STUB1(STIP1 homology and U Box containing protein 1), was identified to be an E3 ligaseassociated with chaperones (19, 20). Many proteins have been reported to be degradedby CHIP through the ubiquitin proteasome pathway, like ErbB2 (21), p53 (22), tau (23)and ER (24). CHIP contains three functional domains: a TPR (tetratricopeptide repeat)domain at the amino terminus, a U box domain at the carboxyl terminus, and a middleregion rich in charged residues. TPR domain is required for binding to CTD(carboxy terminal domain) domain of chaperones (Hsc70 and Hsp90) and U boxdomain is required for its E3 ligase activity. The E3 ligase activity of CHIP isfunctionally linked with Hsc70 and Hsp90 (19, 20). Besides that, it also depends on theUBC4/5 family which belongs to E2 ubiquitin conjugating enzyme. Since the UBC4/5family is stress activated, CHIP may play an important role in converting chaperonecomplexes into a chaperone dependent ubiquitin ligase under stress condition (25). It has been suggested that HIF 1αtends to be degraded in prolonged hypoxia. InHepG2 cells, longer hypoxic treatment (16 hours, 1% O₂) resulted in attenuated HIF 1αprotein expression compared with early hypoxic treatment (5 hours, 1% O₂) (18). InA549 cells, HIF 1αwas similarly stable in acute hypoxia (4 hours, 0.5% O₂), but inprolonged hypoxia, HIF 1αprotein levels decreased (6 to 12 hours, 0.5% O₂) (26). Bothprotein synthesis and degradation are involved in this phenomenon. On the one hand,prolonged hypoxia can inhibit mTOR (Mammalian target of rapamycin) activity, whichleads to low expression levels of HIF 1αby blocking its protein synthesis (27). On theother hand, in prolonged hypoxia transcription activation of HIF 1 complex candecrease its own expression in a negative feedback loop by the VHL/PHD independentway, and acetylation of HIF 1αcan reinforce this degradation (28). Unlike theVHL mediated degradation of HIF 1αin normoxia is widely understood, which E3ligase is responsible for prolonged hypoxia mediated degradation of HIF 1αremains tobe elucidated. In this paper, we report that a known E3 ligase, CHIP targets HIF 1αfordegradation by the ubiquitin proteasome pathway in prolonged hypoxia, which isindependent of VHL and p53/MDM2. Furthermore, acetylation of HIF 1αcan enhanceCHIP induced degradation, which involve in HDAC1 and HDAC2.