Aberrant cytoplasm localization and protein stability of SIRT1 is regulated by PI3K/IGF-1R signaling in human cancer cells

SIRT1, an NAD-dependent histone/protein deacetylase, has classically been thought of as a nuclear protein. In this study, we demonstrate that SIRT1 is mainly localized in the nucleus of normal cells, but is predominantly localized in the cytoplasm of the cancer / transformed cells we tested. We found this predominant cytoplasmic localization of SIRT1 is regulated by elevated mitotic activity and PI3K/IGF-1R signaling in cancer cells. We show that aberrant cytoplasmic localization of SIRT1 is due to increased protein stability and is regulated by PI3K/IGF-1R signaling. In addition, we determined that SIRT1 is required for PI3K-mediated cancer cell growth. Our study represents the first identification that aberrant cytoplasm localization is one of the specific alternations to SIRT1 that occur in cancer cells, and PI3K/IGF-1R signaling plays an important role in the regulation of cytoplasmic SIRT1 stability. Our findings suggest that the over-expressed cytoplasmic SIRT1 in cancer cells may greatly contribute to its cancer-specific function by working downstream of the PI3K/IGF-1R signaling pathway.     

Nuclear transport of H1 histones meets the criteria of a nuclear localization signal—mediated process

We have studied the nuclear transport of H1 histones using the digitonin permeabilization assay system in order to establish the transport requirements for H1 translocation to the nucleus. Using HeLa cells and fluorescence-labeled calf thymus H1, we show that the H1 nuclear transport in permeabilized cells requires the addition of cytoplasmic extract. Furthermore, it can be blocked by energy depletion and by chilling or by addition of wheat germ agglutinin or by nonhydrolyzable GTP analogs. Thus, the import of H1 histones follows the criteria established for nuclear import mediated by nuclear localization signals (NLS). The distribution of basic amino acids in average H1 sequences, however, does not allow the assignment of a specific element as a classical NLS. J. Cell. Biochem. 64:573–578. © 1997 Wiley-Liss, Inc.     

Mapping of the nuclear localization signals in open reading frame 2 protein from porcine circovirus type 1

Porcine circovirus type 1 （PCV1） contains two major open reading frames encoding the replication-associated proteins and the major structural capsid （Cap） protein. PCV1 Cap has an N-terminus carrying several potential monopartite or bipartite nuclear localization signals （NLS）. The contribution of these partially overlapping motifs to nuclear importing was identified by expression of mutated PCVI Cap versions fused to enhanced green fluorescent protein （EGFP）. The Cterminus truncated PCV1 Cap-EGFP was localized in nuclei of PK-15 cells similar to the wild-type PCV1 Cap-EGFP, whereas truncation of the N-terminus rendered the fusion protein distributed into cytoplasm, indicating that the nuclear import of PCV1 Cap was efficiently mediated by its N-terminal region. Substitutions of basic residues in stretches 9RR- RR12 or the right part of 25RRPYLAHPAFRNRYRWRRK43 resulted in a diffused distribution of the fusion protein in both nuclei and cytoplasm, indicating that the two NLSs were responsible for restricted nuclear targeting of PCV1 Cap.     

SIRT5 inhibits peroxisomal ACOX1 to prevent oxidative damage and is downregulated in liver cancer

Peroxisomes account for ~35% of total H₂O₂ generation in mammalian tissues. Peroxisomal ACOX1 (acyl‐CoA oxidase 1) is the first and rate‐limiting enzyme in fatty acid β‐oxidation and a major producer of H₂O₂. ACOX1 dysfunction is linked to peroxisomal disorders and hepatocarcinogenesis. Here, we show that the deacetylase sirtuin 5 (SIRT5) is present in peroxisomes and that ACOX1 is a physiological substrate of SIRT5. Mechanistically, SIRT5‐mediated desuccinylation inhibits ACOX1 activity by suppressing its active dimer formation in both cultured cells and mouse livers. Deletion of SIRT5 increases H₂O₂ production and oxidative DNA damage, which can be alleviated by ACOX1 knockdown. We show that SIRT5 downregulation is associated with increased succinylation and activity of ACOX1 and oxidative DNA damage response in hepatocellular carcinoma (HCC). Our study reveals a novel role of SIRT5 in inhibiting peroxisome‐induced oxidative stress, in liver protection, and in suppressing HCC development.     

EGF-stimulation activates the nuclear localization signal of SHP-1

Protein tyrosine phosphatase SHP-1 plays a critical role in the regulation of a variety of intracellular signaling pathways. SHP-1 is predominantly expressed in the cells of hematopoietic origin, and is recognized as a negative regulator of lymphocyte development and activation. SHP-1 consists of two Src homology 2 (SH2) domains and one protein tyrosine phosphatase (PTP) domain followed by a highly basic C-terminal tail containing tyrosyl phosphorylation sites. It is unclear how the C-terminal tail regulates SHP-1 function. We report the examination of the subcellular localization of a variety of truncated or mutated SHP-1 proteins fused with enhanced green fluorescent protein (EGFP) protein at either the N-terminal or the C-terminal end in different cell lines. Our data demonstrate that a nuclear localization signal (NLS) is located in the C-terminal tail of SHP-1 and the signal is primarily defined by three amino-acid residues (KRK) at the C-terminus. This signal is generally blocked in the native protein and can be exposed by fusing EGFP at the appropriate position or by domain truncation. We have also revealed that this NLS of SHP-1 is triggered by epidermal growth factor (EGF) stimulation and mediates translocation of SHP-1 from the cytosol to the nucleus in COS7 cell lines. These results not only demonstrate the importance of the C-terminal tail of SHP-1 in the regulation of nuclear localization, but also provide insights into its role in SHP-1-involved signal transduction pathways.     

Regulation of COP1 nuclear localization by the COP9 signalosome via direct interaction with CSN1

COP1 and COP9 signalosome (CSN) are key regulators of plant light responses and development. Deficiency in either COP1 or CSN causes a constitutive photomorphogenic phenotype. Through coordinated actions of nuclear- and cytoplasmic-localization signals, COP1 can respond to light signals by differentially partitions between nuclear and cytoplasmic compartments. Previous genetic analysis in Arabidopsis indicated that the nuclear localization of COP1 requires CSN, an eight-subunit heteromeric complex. However the mechanism underlying the functional relationship between COP1 and CSN is unknown. We report here that COP1 weakly associates with CSN in vivo . Furthermore, we report on the direct interaction involving the coiled-coil domain of COP1 and the N-terminal domain of the CSN1 subunit. In onion epidermal cells, expression of CSN1 can stimulate nuclear localization of GUS-COP1, and the N-terminal domain of CSN1 is necessary and sufficient for this function. Moreover, CSN1-induced COP1 nuclear localization requires the nuclear-localization sequences of COP1, as well as its coiled-coil domain, which contains both the cytoplasmic localization sequences and the CSN1 interacting domain. We also provide genetic evidence that the CSN1 N-terminal domain is specifically required for COP1 nuclear localization in Arabidopsis hypocotyl cells. This study advances our understanding of COP1 localization, and the molecular interactions between COP1 and CSN.     

AROS has a context-dependent effect on SIRT1

The modulation of protein deacetylase SIRT1 has a vast therapeutic potential in treatment of several aging-associated diseases. Active regulator of SIRT1 (AROS) is a small endogenous protein which was originally reported to activate SIRT1 through a direct interaction in cancer cells. We show that the interaction between the two proteins is weak and does not alter the activity of SIRT1 in non-cancerous human cells. The results of different in vitro SIRT1 activity assays disclosed AROS as an inhibitor of SIRT1. The functional relationship between AROS and SIRT1 proved to be dependent on the biological context and experimental setting.     

Extracellular NAMPT/visfatin causes p53 deacetylation via NAD production and SIRT1 activation in breast cancer cells

Visfatin, which is secreted as an adipokine and cytokine, has been implicated in cancer development and progression. In this study, we investigated the NAD‐producing ability of visfatin and its relationship with SIRT1 (silent information regulator 2) and p53 to clarify the role of visfatin in breast cancer. MCF‐7 breast cancer cells were cultured and treated with visfatin. SIRT1 activity was assessed by measuring fluorescence intensity from fluoro‐substrate peptide. To investigate the effect of visfatin on p53 acetylation, SDS‐PAGE followed by western blotting was performed using specific antibodies against p53 and its acetylated form. Total NAD was measured both in cell lysate and the extracellular medium by colorimetric method. Visfatin increased both extracellular and intracellular NAD concentrations. It also induced proliferation of breast cancer cells, an effect that was abolished by inhibition of its enzymatic activity. Visfatin significantly increased SIRT1 activity, accompanied by induction of p53 deacetylation. In conclusion, the results show that extracellular visfatin produces NAD that causes upregulation of SIRT1 activity and p53 deacetylation. These findings explain the relationship between visfatin and breast cancer progression.     

SIRT1 activation by SRT1720 attenuates bone cancer pain via preventing Drp1-mediated mitochondrial fission

Bone cancer pain (BCP) is the pain induced by primary bone cancer or tumor metastasis. Increasing evidence and our previous studies have shown that mammalian silent information regulator 2?homolog (SIRT1) is involved in periphery sensitization and central sensitization of BCP, and the underlying mechanism of SIRT1 in bone cancer pain may provide clues for pain treatment. Dynamin-related protein 1 (Drp1) is an essential regulator for mitochondrial fission. In this research, BCP model rats were established by injecting MRMT-1 rat mammary gland carcinoma cells into the left tibia of female Sprague-Dawley rats and validated by tibia radiographs, histological examination and mechanical pain test. As a result BCP rats exhibited bone destruction and sensitivity mechanical pain. BCP increased inflammatory cells infiltration and apoptosis, reduced SIRT1 protein expression and phosphorylation, and elevated Drp1 expression in spinal cord. An agonist of SIRT1 named SRT1720 intrathecal treatment in BCP rats increased SIRT1 phosphorylation, reduced the up-regulated Drp1 expression, and reversed pain behavior. SRT1720 also regulated Bcl-2/BAX and cleaved caspase-3 expressions, and inhibited mitochondrial apoptosis in spinal cord of BCP rats. For in vitro research, SRT1720 treatment decreased Drp1 expression in a dose-dependent manner, blocked CCCP-induced mitochondrial membrane potential change, consequently reduced apoptosis and promoted proliferation. These data suggest that SIRT1 activation by SRT1720 attenuated bone cancer pain via preventing Drp1-mediated mitochondrial fission. Our results provide new targets for therapeutics of bone cancer pain.     

Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells

Overexpression of SIRT1, a NAD⁺-dependent class III histone deacetylases (HDACs), is implicated in many cancers and therefore could become a promising antitumor target. Here we demonstrate a small molecule SIRT1 inhibitor, ILS-JGB-1741(JGB1741) with potent inhibitory effects on the proliferation of human metastatic breast cancer cells, MDA-MB 231. The molecule has been designed using medicinal chemistry approach based on known SIRT1 inhibitor, sirtinol. The molecule showed a significant inhibition of SIRT1 activity compared to sirtinol. Studies on the antitumor effects of JGB on three different cancer cell lines, K562, HepG2 and MDA-MB 231 showed an IC₅₀ of 1, 10 and 0.5 μM, respectively. Further studies on MDA-MB 231 cells showed a dose-dependent increase in K9 and K382 acetylation of H3 and p53, respectively. Results also demonstrated that JGB1741-induced apoptosis is associated with increase in cytochrome c release, modulation in Bax/Bcl2 ratio and cleavage of PARP. Flowcytometric analysis showed increased percentage of apoptotic cells, decrease in mitochondrial membrane potential and increase in multicaspase activation. In conclusion, the present study indicates the potent apoptotic effects of JGB1741 in MDA-MB 231 cells.     

The NPK1 mitogen-activated protein kinase kinase kinase contains a functional nuclear localization signal at the binding site for the NACK1 kinesin-like protein

The tobacco mitogen-activated protein kinase kinase kinase NPK1 localizes to the equatorial region of phragmoplasts by interacting with kinesin-like protein NACK1. This leads to activation of NPK1 kinase at late M phase, which is necessary for cell plate formation. Until now, its localization during interphase has not been reported. We investigated the subcellular localization of NPK1 in tobacco-cultured BY-2 cells at interphase using indirect immunofluorescence microscopy and fusion to green fluorescent protein (GFP). Fluorescence of anti-NPK1 antibodies and GFP-fused NPK1 were detected only in the nuclei of BY-2 cells at interphase. Examination of the amino acid sequence of NPK1 showed that at the carboxyl-terminal region in the regulatory domain, which contains the binding site of NACK1, NPK1 contained a cluster of basic amino acids that resemble a bipartite nuclear localization signal (NLS). Amino acid substitution mutations in the critical residues in putative NLS caused a marked reduction in nuclear localization of NPK1 in BY-2 cells, indicating that this sequence is functional in tobacco BY-2 cells. We also found that the 64-amino acid sequence at the carboxyl terminus that contains NLS sequence is essential for interaction with NACK1, and that mutations in the NLS sequence prevented NPK1 from interacting with NACK1. Thus, the amino acid sequence at the carboxyl-terminal region of NPK1 has dual functions for nuclear localization during interphase and binding NACK1 in M phase.     

Methionine metabolism is essential for SIRT1‐regulated mouse embryonic stem cell maintenance and embryonic development

Methionine metabolism is critical for epigenetic maintenance, redox homeostasis, and animal development. However, the regulation of methionine metabolism remains unclear. Here, we provide evidence that SIRT1, the most conserved mammalian NAD⁺‐dependent protein deacetylase, is critically involved in modulating methionine metabolism, thereby impacting maintenance of mouse embryonic stem cells (mESCs) and subsequent embryogenesis. We demonstrate that SIRT1‐deficient mESCs are hypersensitive to methionine restriction/depletion‐induced differentiation and apoptosis, primarily due to a reduced conversion of methionine to S‐adenosylmethionine. This reduction markedly decreases methylation levels of histones, resulting in dramatic alterations in gene expression profiles. Mechanistically, we discover that the enzyme converting methionine to S‐adenosylmethionine in mESCs, methionine adenosyltransferase 2a (MAT2a), is under control of Myc and SIRT1. Consistently, SIRT1 KO embryos display reduced Mat2a expression and histone methylation and are sensitive to maternal methionine restriction‐induced lethality, whereas maternal methionine supplementation increases the survival of SIRT1 KO newborn mice. Our findings uncover a novel regulatory mechanism for methionine metabolism and highlight the importance of methionine metabolism in SIRT1‐mediated mESC maintenance and embryonic development.     

Expression of cancer related BRCA1 missense variants decreases MMS-induced recombination in Saccharomyces cerevisiae without altering its nuclear localization

BRCA1 tumor suppressor gene is found mutated in familial breast and ovarian cancer. Most cancer related mutations were found located at the RING (Really Interesting New Gene) and at the BRCT (BRca1 C-Terminal) domain. However, 20 y after its identification, the biological role of BRCA1 and which domains are more relevant for tumor suppression are still being elucidated. We previously reported that expression of BRCA1 cancer related variants in the RING and BRCT domain increases spontaneous homologous recombination in yeast indicating that BRCA1 may interact with yeast DNA repair/recombination. To finally demonstrate whether BRCA1 interacts with yeast DNA repair, we exposed yeast cells expressing BRCA1wt, the cancer-related variants C-61G and M1775R to different doses of the alkylating agent methyl methane-sulfonate (MMS) and then evaluated the effect on survival and homologous recombination. Cells expressing BRCA1 cancer variants were more sensitive to MMS and less inducible to recombination as compared to cell expressing BRCA1wt. Moreover, BRCA1-C61G and -M1775R did not change their nuclear localization form as compared to the BRCA1wt or the neutral variant R1751Q indicating a difference in the DNA damage processing. We propose a model where BRCA1 cancer variants interact with the DNA double strand break repair pathways producing DNA recombination intermediates, that maybe less repairable and decrease MMS-induced recombination and survival. Again, this study strengthens the use of yeast as model system to characterize the mechanisms leading to cancer in humans carrying the BRCA1 missense variant.