II-6

Amino Acid Sequence

3D Structure (/structures/AF-Q8IXJ6-F1-model_v6.cif)

Function

NAD-dependent protein deacetylase, which deacetylates internal lysines on histone and alpha-tubulin as well as many other proteins such as key transcription factors. Participates in the modulation of multiple and diverse biological processes such as cell cycle control, genomic integrity, microtubule dynamics, cell differentiation, metabolic networks, and autophagy. Plays a major role in the control of cell cycle progression and genomic stability. Functions in the antephase checkpoint preventing precocious mitotic entry in response to microtubule stress agents, and hence allowing proper inheritance of chromosomes. Positively regulates the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase complex activity by deacetylating CDC20 and FZR1, then allowing progression through mitosis. Associates both with chromatin at transcriptional start sites (TSSs) and enhancers of active genes. Plays a role in cell cycle and chromatin compaction through epigenetic modulation of the regulation of histone H4 'Lys-20' methylation (H4K20me1) during early mitosis. Specifically deacetylates histone H4 at 'Lys-16' (H4K16ac) between the G2/M transition and metaphase enabling H4K20me1 deposition by KMT5A leading to ulterior levels of H4K20me2 and H4K20me3 deposition throughout cell cycle, and mitotic S-phase progression. Deacetylates KMT5A modulating KMT5A chromatin localization during the mitotic stress response. Also deacetylates histone H3 at 'Lys-57' (H3K56ac) during the mitotic G2/M transition. Upon bacterium Listeria monocytogenes infection, deacetylates 'Lys-18' of histone H3 in a receptor tyrosine kinase MET- and PI3K/Akt-dependent manner, thereby inhibiting transcriptional activity and promoting late stages of listeria infection. During oocyte meiosis progression, may deacetylate histone H4 at 'Lys-16' (H4K16ac) and alpha-tubulin, regulating spindle assembly and chromosome alignment by influencing microtubule dynamics and kinetochore function. Deacetylates histone H4 at 'Lys-16' (H4K16ac) at the VEGFA promoter and thereby contributes to regulate expression of VEGFA, a key regulator of angiogenesis. Deacetylates alpha-tubulin at 'Lys-40' and hence controls neuronal motility, oligodendroglial cell arbor projection processes and proliferation of non-neuronal cells. Phosphorylation at Ser-368 by a G1/S-specific cyclin E-CDK2 complex inactivates SIRT2-mediated alpha-tubulin deacetylation, negatively regulating cell adhesion, cell migration and neurite outgrowth during neuronal differentiation. Deacetylates PARD3 and participates in the regulation of Schwann cell peripheral myelination formation during early postnatal development and during postinjury remyelination. Involved in several cellular metabolic pathways. Plays a role in the regulation of blood glucose homeostasis by deacetylating and stabilizing phosphoenolpyruvate carboxykinase PCK1 activity in response to low nutrient availability. Acts as a key regulator in the pentose phosphate pathway (PPP) by deacetylating and activating the glucose-6-phosphate G6PD enzyme, and therefore, stimulates the production of cytosolic NADPH to counteract oxidative damage. Maintains energy homeostasis in response to nutrient deprivation as well as energy expenditure by inhibiting adipogenesis and promoting lipolysis. Attenuates adipocyte differentiation by deacetylating and promoting FOXO1 interaction to PPARG and subsequent repression of PPARG-dependent transcriptional activity. Plays a role in the regulation of lysosome-mediated degradation of protein aggregates by autophagy in neuronal cells. Deacetylates FOXO1 in response to oxidative stress or serum deprivation, thereby negatively regulating FOXO1-mediated autophagy. Deacetylates a broad range of transcription factors and co-regulators regulating target gene expression. Deacetylates transcriptional factor FOXO3 stimulating the ubiquitin ligase SCF(SKP2)-mediated FOXO3 ubiquitination and degradation. Deacetylates HIF1A and therefore promotes HIF1A degradation and inhibition of HIF1A transcriptional activity in tumor cells in response to hypoxia. Deacetylates RELA in the cytoplasm inhibiting NF-kappaB-dependent transcription activation upon TNF stimulation. Inhibits transcriptional activation by deacetylating p53/TP53 and EP300. Also deacetylates EIF5A. In addition to protein deacetylase activity, also acts as a protein-lysine deacylase by recognizing other acyl groups: catalyzes removal of N6-benzoyl (benzoyl) and N6-methacryl (methacryl) acyl groups from lysine residues, leading to histone debenzoylation and demethacrylation, respectively. Functions as a negative regulator on oxidative stress-tolerance in response to anoxia-reoxygenation conditions. Plays a role as tumor suppressor. In addition to protein deacetylase activity, also has activity toward long-chain fatty acyl groups and mediates protein-lysine demyristoylation and depalmitoylation of target proteins, such as ARF6 and KRAS, thereby regulating their association with membranes.

Mechanism

The autophagy-lysosomal pathway (ALP) is capable of degrading a broader range of substrates, including longer-lived and larger misfolded proteins and damaged organelles. ALP is classified into three subtypes: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. In microautophagy, substrates are directly sequestered and degraded by lysosomes and late endosomes via membrane protrusion and invagination. In CMA, HSPA8/HSC70 recognizes specific substrates containing KFERQ-like sequences; these substrates subsequently bind to LAMP2A on the lysosomal surface and are delivered into lysosomes for degradation. In macroautophagy, substrates are encapsulated through the formation of double-membrane autophagosomes, which subsequently fuse with lysosomes to degrade the substrates.

Description

To further explore the intricate mechanism of II-6 induced SIRT2 degradation, the SIRT2 degradation was examined by blocking the ubiquitin proteasome pathway and autophagolysosomal pathway with l -tryptophanyl- l -alanine (H-TRP-ALA-OH), chloroquine, and 3-methyladenine (3-MA), respectively. The results showed that the intervention of tenovin-6 can partly prevent the degradation of SIRT2 ( Fig. 1 J), which suggests that the affinity with SIRT2 is essential for the degradation. As expected, SIRT2 protein degradation induced by II-6 could be effectively blocked and reversed with proteasome inhibitors (H-TRP-ALA-OH) and autophagy inhibitors (3MA and chloroquine). The results indicated that II-6 -induced SIRT2 protein degradation was not only UPS dependent, but autophagy-lysosome pathway as well.

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

To further explore the intricate mechanism of II-6 induced SIRT2 degradation, the SIRT2 degradation was examined by blocking the ubiquitin proteasome pathway and autophagolysosomal pathway with l -tryptophanyl- l -alanine (H-TRP-ALA-OH), chloroquine, and 3-methyladenine (3-MA), respectively. The results showed that the intervention of tenovin-6 can partly prevent the degradation of SIRT2 ( Fig. 1 J), which suggests that the affinity with SIRT2 is essential for the degradation. As expected, SIRT2 protein degradation induced by II-6 could be effectively blocked and reversed with proteasome inhibitors (H-TRP-ALA-OH) and autophagy inhibitors (3MA and chloroquine). The results indicated that II-6 -induced SIRT2 protein degradation was not only UPS dependent, but autophagy-lysosome pathway as well.

Description

The degraders were firstly evaluated for their anti-proliferative activity against OC cells, including SKOV3, OVCAR3, A2780 and ovarian epithelial cell (IOSE80 cell) as well using an in vitro cell counting kit-8 (CCK-8) assay. In particular, most of the compounds exhibit superior inhibitory effects in the A2780 cells compared to the OVCAR3 and SKOV3 cells. Particularly, the compounds I-1–I-6 , tagged with amantadine, were more effective to inhibit proliferation of cancer cells. Among them, compound I-6 containing ten carbon-atom linker achieved an half maximal inhibitory concentration (IC50 ) value of 0.008 μmol/L which was much lower than that of cisplatin (IC50 = 1.28 μmol/L). However, length of linker had no obvious effect on the anti-tumor activity of compounds I in vitro . We found that the replacement of amantadine with Boc1 -Trp such as II-6 increased potency accordingly (IC50 = 0.002 ±0.001 μmol/L).