The deubiquitinating enzyme USP4 regulates BRCA1 stability and function.

BRCA1 plays a suppressive role in breast tumorigenesis. Ubiquitin-dependent degradation is a common mechanism that regulates BRCA1 protein stability, and several ubiquitin ligases involved have been identified. However, the deubiquitinating enzyme for BRCA1 remains less defined. Here, we report that the deubiquitinase USP4 interacts with, deubiquitinates and stabilizes BRCA1, maintaining the protein level of BRCA1. USP4 knockdown results in a decreased BRCA1 protein level, impairment in homologous recombination mediated double-stranded break repair, and increased genome instability, and confers resistance to DNA damage-inducing agents and PARP inhibitors. Ectopic expression of USP4 stabilizes BRCA1 and reverse the effects caused by USP4 knockdown. Moreover, USP4 is low expressed in human breast cancer tissues and its low expression correlates with poorer survival of patients. Furthermore, we identified several loss-of-function mutations of USP4 in human gynecological cancers, the catalytic activity of which or their interaction with BRCA1 is disrupted. Together, we reveal that USP4 is a deubiquitinase for BRCA1. USP4 positively regulates the stability and function of BRCA1 through de-ubiquitination, and plays important role in the suppression of breast cancer.

Cisplatin-activated ERß/DCAF8 positive feedback loop induces chemoresistance in non-small cell lung cancer via PTEN/Akt axis.

High levels of the estrogen receptor ß (ERß) predict poor prognosis following platinum-containing adjuvant chemotherapies in patients with non-small cell lung cancer (NSCLC). However, the precise role of ERß remains elusive. In this study, we demonstrated that targeting ERß could significantly increase the cytotoxicity of cisplatin both in vitro and in vivo. Mechanically, cisplatin directly binds to ERß, which facilitates its homodimerization and nuclear translocation. ERß activation transcriptionally represses the expression of DCAF8, an adaptor of CRL4 E3 ubiquitin ligase, which in turn attenuates the proteasomal degradation of ERß, leading to ERß accumulation; this positive feedback loop results in Akt activation and eventually cisplatin resistance in NSCLC through PTEN inhibition. Moreover, low expression of DCAF8 and high expression of ERß are associated with treatment resistance in patients receiving cisplatin-containing adjuvant chemotherapy. The present results provide insights into the underlying mechanism of ERß-induced cisplatin resistance and offer an alternative therapeutic strategy to improve the efficacy of platinum-based chemotherapy in patients with NSCLC.

USP11-mediated LSH deubiquitination inhibits ferroptosis in colorectal cancer through epigenetic activation of CYP24A1.

Ferroptosis is an iron-dependent form of regulated cell death characterized by lipid peroxidation. Colorectal cancer (CRC) cells evade ferroptosis despite their requirement of substantial iron and reactive oxygen species (ROS) to sustain active metabolism and extensive proliferation. However, the underlying mechanism is unclear. Herein, we report the role of lymphoid-specific helicase (LSH), a chromatin-remodeling protein, in suppressing erastin-induced ferroptosis in CRC cells. We demonstrate that erastin treatment leads to dose- and time-dependent downregulation of LSH in CRC cells, and depletion of LSH increases cell sensitivity to ferroptosis. Mechanistically, LSH interacts with and is stabilized by ubiquitin-specific protease 11 (USP11) via deubiquitination; this interaction was disrupted by erastin treatment, resulting in increased ubiquitination and LSH degradation. Moreover, we identified cytochrome P450 family 24 subfamily A member 1 (CYP24A1) as a transcriptional target of LSH. LSH binds to the CYP24A1 promoter, promoting nucleosome eviction and reducing H3K27me3 occupancy, thus leading to transcription of CYP24A1. This cascade inhibits excessive intracellular Ca2+ influx, thereby reducing lipid peroxidation and ultimately conferring resistance to ferroptosis. Importantly, aberrant expression of USP11, LSH, and CYP24A1 is observed in CRC tissues and correlates with poor patient prognosis. Taken together, our study demonstrates the crucial role of the USP11/LSH/CYP24A1 signaling axis in inhibiting ferroptosis in CRC, highlighting its potential as a therapeutic target in CRC treatment.

The deubiquitinating enzyme complex BRISC regulates Aurora B activation via lysine-63-linked ubiquitination in mitosis.

Faithful chromosome segregation requires bi-oriented kinetochore-microtubule attachment on the metaphase spindle. Aurora B kinase, the catalytic core of the chromosome passage complex (CPC), plays a crucial role in this process. Aurora B activation has widely been investigated in the context of protein phosphorylation. Here, we report that Aurora B is ubiquitinated in mitosis through lysine-63 ubiquitin chains (K63-Ub), which is required for its activation. Mutation of Aurora B at its primary K63 ubiquitin site inhibits its activation, reduces its kinase activity, and disrupts the association of Aurora B with other components of CPC, leading to severe mitotic defects and cell apoptosis. Moreover, we identify that BRCC36 isopeptidase complex (BRISC) is the K63-specific deubiquitinating enzyme for Aurora B. BRISC deficiency augments the accumulation of Aurora B K63-Ubs, leading to Aurora B hyperactivation and erroneous chromosome-microtubule attachments. These findings define the role of K63-linked ubiquitination in regulating Aurora B activation and provide a potential site for Aurora B-targeting drug design.

CRL4-DCAF8L2 E3 ligase promotes ubiquitination and degradation of BARD1.

BARD1 is a tumor suppressor that is necessary for the functioning and stability of BRCA1, with which it forms a heterodimer and participates in the repair of DNA double-strand breaks. The cellular level of BARD1 and its interaction with BRCA1 are crucial for BRCA1/BARD1 function in homologous recombination and tumor suppression. However, the regulatory mechanism underpinning the stability of BARD1 is largely unclear. In this study, we identified DCAF8L2, a DDB1-Cullin associated factor (DCAF) associated with CRL4 E3 ligase, as a negative regulator of BARD1. Mechanistically, DCAF8L2 interacts with and targets BARD1 for ubiquitination and degradation. In addition, the interaction of DCAF8L2 with BARD1 through the RING domain could compete with the dimerization of BRCA1 and BARD1, leading to increased cellular uncoupling of BARD1 and BRCA1, subjecting the latter to degradation. The overexpression of DCAF8L2 compromises the homologous recombination process and confers cells with increased sensitivity to DNA damage. Furthermore, DCAF8L2 was aberrantly expressed in breast cancer cell lines. Our findings suggest that DCAF8L2 may play an oncogenic role in the pathogenesis of breast cancer, possibly by negative regulation of BARD1.

CRL4-DCAF8L1 Regulates BRCA1 and BARD1 Protein Stability.

BRCA1 is frequently down-regulated in breast cancer, the underlying mechanism is unclear. Here we identified DCAF8L1, an X-linked gene product, as a DDB1-Cullin associated Factor (DCAF) for CUL4 E3 ligases to target BRCA1 and BARD1 for proteasomal degradation. Forced expression of DCAF8L1 caused reduction of BRCA1 and BARD1, and impaired DNA damage repair function, conferring increased sensitivity to irradiation and DNA damaging agents, as well as Olaparib, a PARPi anticancer drug; while depletion of DCAF8L1 restored BRCA1 and suppressed the growth of its xenograft tumors. Furthermore, the expression of DCAF8L1 was induced in human H9 ES cells during transition from primed to naïve state when Xi chromosome was reactivated. Aberrant expression of DCAF8L1 was observed in human breast fibroadenoma and breast cancer. These findings suggest that CRL4DCAF8L1 is an important E3 ligase that may participate in the development of breast cancer, probably through regulating the stability of BRCA1 and BARD1 tumor suppressor, linking BRCA1 and X chromosome inactivation to breast carcinogenesis.

RNF8-mediated regulation of Akt promotes lung cancer cell survival and resistance to DNA damage.

Despite the tremendous success of targeted and conventional therapies for lung cancer, therapeutic resistance is a common and major clinical challenge. RNF8 is a ubiquitin E3 ligase that plays essential roles in the DNA damage response; however, its role in the pathogenesis of lung cancer is unclear. Here, we report that RNF8 is overexpressed in lung cancer and positively correlates with the expression of p-Akt and poor survival of patients with non-small-cell lung cancer. In addition, we identify RNF8 as the E3 ligase for regulating the activation of Akt by K63-linked ubiquitination under physiological and genotoxic conditions, which leads to lung cancer cell proliferation and resistance to chemotherapy. Together, our study suggests that RNF8 could be a very promising target in precision medicine for lung cancer.

Dezocine, An Opioid Analgesic, Exerts Antitumor Effects in Triple-Negative Breast Cancer by Targeting Nicotinamide Phosphoribosyltransferase.

Opioids are a potential adjuvant treatment for certain cancers; while they are primarily used to relieve chronic pain, these drugs may also affect cancer progression and recurrence. Dezocine is one opioid commonly used in China, but its effects on cancer cells are unknown. Here, we demonstrated the inhibitory effect of dezocine on triple-negative breast cancer (TNBC) cells, and determined the underlying molecular mechanism. We found that dezocine suppressed cell proliferation, migration and invasion, and induced apoptosis in TNBC cells. Xenograft models demonstrated the inhibitory effects of dezocine treatment on TNBC tumor growth in vivo. The anticancer effects of dezocine were independent of opioid receptors, which are not highly expressed by normal breast or breast cancer tissues. A pull-down assay and LC-MS/MS analysis indicated that dezocine directly targets NAMPT: computer modeling verified that the free energy of dezocine kinetically bound into the pocket of NAMPT was -17.4 kcal/mol. Consequently, dezocine treatment inhibited NAMPT enzyme activity, resulting in cellular NAD abolishment. We confirmed the dezocine-induced inhibition of cell proliferation by both NAMPT knockdown and upon treatment with the inhibitor FK866. Our results suggest that both dezocine and NAMPT might represent novel therapeutic targets for TNBC.

The deubiquitinase USP11 regulates cell proliferation and ferroptotic cell death via stabilization of NRF2 USP11 deubiquitinates and stabilizes NRF2.

The transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) plays a key role in cancer progression and is tightly regulated by the proteasome pathway. E3 ligases that mediate NRF2 ubiquitination have been widely reported, but the mechanism of NRF2 deubiquitination remains largely unclear. Here, we identified ubiquitin-specific-processing protease 11 (USP11) in NRF2 complexes and confirmed an interaction between these two proteins. We further found that USP11 deubiquitinates NRF2; this modification stabilizes NRF2. Functionally, USP11 depletion contributes to the suppression of cell proliferation and induction of ferroptotic cell death due to ROS-mediated stress, which can be largely abrogated by overexpression of NRF2. Finally, immunohistochemical staining of USP11 and NRF2 was performed using a lung tissue microarray, which revealed that USP11 is highly expressed in patients with NSCLC and positively correlated with NRF2 expression. Together, USP11 stabilizes NRF2 and is thus an important player in cell proliferation and ferroptosis.

HUWE1 mediates inflammasome activation and promotes host defense against bacterial infection.

The mechanism by which inflammasome activation is modulated remains unclear. In this study, we identified an AIM2-interacting protein, the E3 ubiquitin ligase HUWE1, which was also found to interact with NLRP3 and NLRC4 through the HIN domain of AIM2 and the NACHT domains of NLRP3 and NLRC4. The BH3 domain of HUWE1 was important for its interaction with NLRP3, AIM2, and NLRC4. Caspase-1 maturation, IL-1ß release, and pyroptosis were reduced in Huwe1-deficient bone marrow-derived macrophages (BMDMs) compared with WT BMDMs in response to stimuli to induce NLRP3, NLRC4, and AIM2 inflammasome activation. Furthermore, the activation of NLRP3, NLRC4, and AIM2 inflammasomes in both mouse and human cells was remarkably reduced by treatment with the HUWE1 inhibitor BI8622. HUWE1 mediated the K27-linked polyubiquitination of AIM2, NLRP3, and NLRC4, which led to inflammasome assembly, ASC speck formation, and sustained caspase-1 activation. Huwe1-deficient mice had an increased bacterial burden and decreased caspase-1 activation and IL-1ß production upon Salmonella, Francisella, or Acinetobacter baumannii infection. Our study provides insights into the mechanisms of inflammasome activation as well as a potential therapeutic target against bacterial infection.

Citrate synthase desuccinylation by SIRT5 promotes colon cancer cell proliferation and migration.

Citrate synthase (CS), the rate-limiting enzyme in the tricarboxylic acid (TCA) cycle catalyzes the first step of the cycle, namely, the condensation of oxaloacetate and acetyl-CoA to produce citrate. The expression and enzymatic activity of CS are altered in cancers, but posttranslational modification (PTM) of CS and its regulation in tumorigenesis remain largely obscure. SIRT5 belongs to the nicotinamide adenine dinucleotide (NAD)+-dependent deacetylase sirtuin family and plays vital roles in multiple biological processes via modulating various substrates. Here, we show that SIRT5 interacts with CS and that SIRT5 desuccinylates CS at the evolutionarily conserved residues K393 and K395. Moreover, hypersuccinylation of CS at K393 and K395 dramatically reduces its enzymatic activity and suppresses colon cancer cell proliferation and migration. These results provide experimental evidence in support of a potential therapeutic approach for colon cancer.

RXXPEG motif of MERIT40 is required to maintain spindle structure and function through its interaction with Tankyrase1.

Deubiquitinase BRISC complex plays important role in the maintenance of spindle structure and function; however, the underlying mechanism remains largely undefined. Here we demonstrated that MERIT40, a core component of BRISC complex, directly interacts with the RXXPEG motif in the ARC-V domain of Tankyrase1(TNKS1). Mutation of the RXXPEG motif in the MERIT40 (R28A) disrupted its interaction with TNKS1. Consistent with these data, R28A mutant cells displayed multiple mitotic defects including aberrant spindle assembly and chromosome misalignment. These results support a critical role of RXXPEG motif of MERIT40 in BRISC-mediated regulation of TNKS1 function during spindle assembly.

CRL4BRBBP7 targets HUWE1 for ubiquitination and proteasomal degradation.

The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in diverse biological processes including DNA damage repair and apoptosis. Our previous study has shown that in response to DNA damage HUWE1 was downregulated in CUL4B-mediated ubiquitination and subsequent proteasomal degradation, and CUL4B-mediated regulation of HUWE1 was important for cell survival upon DNA damage. CUL4B is a core component of the CUL4B Ring ligase complexes containing ROC1, DDB1 and a DDB1-Cullin Associated Factors (DCAFs), the latter of which are DDB1-binding WD40 adaptors critical for substrate recognition and recruitment. However, the identity of DCAF in CRL4B that mediates degradation of HUWE1 remains elusive. Here we report that RBBP7 is the DCAF in the CRL4B complex bridging the DDB1-CUL4B-ROC1 to HUWE1. Loading of HUWE1 to the E3 ubiquitin ligase complex resulted in its polyubiquitination, and consequently its proteasome mediated degradation. Overexpression of RBBP7 promoted HUWE1 protein degradation, while depletion of RBBP7 stabilized HUWE1, and hence accelerated the degradation of MCL-1 and BRCA1, two substrates of HUWE1 that are critical in apoptosis and DNA damage repair. Taken together, these data reveal CRL4BRBBP7 is the E3 ligase responsible for the proteasomal degradation of HUWE1, and further provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase complex.

The E3 ubiquitin ligase c-Cbl mediates integrin ß1 ubiquitination during dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by dilatation of the ventricular chambers and impaired myocardial contractility. The results of our previous study indicated that a deficiency in matricellular cartilage oligomeric matrix protein (COMP) led to spontaneous and progressive DCM in mice via the ubiquitination/degradation of integrin ß1. However, the specific ubiquitin enzyme involved in degradation of integrin ß1 and the pathogenesis of DCM remain elusive. We first compared gene expression profiles in hearts from 3-month-old wild type and COMP-/- mice using microarray analysis. Among the E3 ubiquitin ligases upregulated in COMP-/- hearts, c-Cbl silencing rescued the ubiquitination/degradation of integrin ß1, myofilament loss, apoptosis and connexin-43 deficiency in cardiomyocytes due to the silencing of COMP. Furthermore, c-Cbl silencing by intramyocardial injections of siRNA into 1-month-old COMP-/- mice ameliorated spontaneous DCM in vivo, as evidenced by the inhibition of the dilation of ventricular chambers, impaired ejection fraction and myofilament loss. A subsequent cellular ubiquitination assay revealed that overexpression of c-Cbl induced ubiquitination of integrin ß1, whereas the G306E mutation in c-Cbl, which prevented the binding of c-Cbl to its substrates, had no effect on integrin ß1 ubiquitination, indicating that c-Cbl directly caused the ubiquitination of integrin ß1 in the hearts. In conclusion, our results demonstrate that c-Cbl mediates the ubiquitination/degradation of integrin ß1, which leads to COMP deficiency-induced DCM.

RNF8 promotes epithelial-mesenchymal transition of breast cancer cells.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is a crucial step for solid tumor progression and plays an important role in cancer invasion and metastasis. RNF8 is an ubiquitin E3 ligase with RING domain, and plays essential roles in DNA damage response and cell cycle regulation. However the role of RNF8 in the pathogenesis of breast cancer is still unclear. METHODS: The expression of RNF8 was examined in different types of breast cell lines by Western Blotting. EMT associated markers were examined by Immunofluorescence and Western Blotting in MCF-7 when RNF8 was ectopically overexpressed, or in MDA-MB-231 when RNF8 was depleted. Transwell and wound healing assays were performed to assess the effect of RNF8 on cell mobility. The xenograft model was done with nude mice to investigate the role of RNF8 in tumor metastasis in vivo. Breast tissue arrays were used to examine the expression of RNF8 by immunohistochemistry. Kaplan-Meier survival analysis for the relationship between survival time and RNF8 signature in breast cancer was done with an online tool ( http://kmplot.com/analysis/ ). RESULTS: RNF8 is overexpressed in highly metastatic breast cancer cell lines. Overexpression of RNF8 in MCF-7 significantly promoted EMT phenotypes and facilitated cell migration. On the contrary, silencing of RNF8 in MDA-MB-231 induced MET phenotypes and inhibited cell migration. Furthermore, we proved that these metastatic behavior promoting effects of RNF8 in breast cancer was associated with the inactivation of GSK-3ß and activation of ß-catenin signaling. With nude mice xenograft model, we found that shRNA mediated-downregulation of RNF8 reduced tumor metastasis in vivo. In addition, we found that RNF8 expression was higher in malignant breast cancer than that of the paired normal breast tissues, and was positively correlated with lymph node metastases and poor survival time. CONCLUSIONS: RNF8 induces EMT in the breast cancer cells and promotes breast cancer metastasis, suggesting that RNF8 could be used as a potential therapeutic target for the prevention and treatment of breast cancer.

The deubiquitinating enzyme complex BRISC is required for proper mitotic spindle assembly in mammalian cells.

Deubiquitinating enzymes (DUBs) negatively regulate protein ubiquitination and play an important role in diverse physiological processes, including mitotic division. The BRCC36 isopeptidase complex (BRISC) is a DUB that is specific for lysine 63-linked ubiquitin hydrolysis; however, its biological function remains largely undefined. Here, we identify a critical role for BRISC in the control of mitotic spindle assembly in cultured mammalian cells. BRISC is a microtubule (MT)-associated protein complex that predominantly localizes to the minus ends of K-fibers and spindle poles and directly binds to MTs; importantly, BRISC promotes the assembly of functional bipolar spindle by deubiquitinating the essential spindle assembly factor nuclear mitotic apparatus (NuMA). The deubiquitination of NuMA regulates its interaction with dynein and importin-ß, which are required for its function in spindle assembly. Collectively, these results uncover BRISC as an important regulator of the mitotic spindle assembly and cell division, and have important implications for the development of anticancer drugs targeting BRISC.

DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation.

The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in integrating/coordinating diverse cellular processes such as DNA damage repair and apoptosis. A previous study has shown that HUWE1 is required for the early step of DNA damage-induced apoptosis, by targeting MCL-1 for proteasomal degradation. However, HUWE1 is subsequently inactivated, promoting cell survival and the subsequent DNA damage repair process. The mechanism underlying its regulation during this process remains largely undefined. Here, we show that the Cullin4B-RING E3 ligase (CRL4B) is required for proteasomal degradation of HUWE1 in response to DNA damage. CUL4B is activated in a NEDD8-dependent manner, and ubiquitinates HUWE1 in vitro and in vivo. The depletion of CUL4B stabilizes HUWE1, which in turn accelerates the degradation of MCL-1, leading to increased induction of apoptosis. Accordingly, cells deficient in CUL4B showed increased sensitivity to DNA damage reagents. More importantly, upon CUL4B depletion, these phenotypes can be rescued through simultaneous depletion of HUWE1, consistent with the role of CUL4B in regulating HUWE1. Collectively, these results identify CRL4B as an essential E3 ligase in targeting the proteasomal degradation of HUWE1 in response to DNA damage, and provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase.

HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin-proteasome pathway.

The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn-proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1 binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin-proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.

HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin-proteasome pathway.

The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn-proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin-proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.

Argininosuccinate synthetase gene is silenced by CpG methylation in children with phenylketonuria.

OBJECTIVES: The concentration of tyrosine and the ratio of branch-amino acid to the aromatic amino acid in phenylketonuria (PKU) patients are much lower than that of normal people, which reveal that PKU patients have amino acid metabolism disorder. The aim of the present study was to investigate the arginine level in blood, the expression of argininosuccinate synthetase (ASS), the rate-limiting enzyme in arginine synthesis pathway, and the methylation of ASS in patients with PKU. DESIGN AND METHODS: Twenty-five children with PKU and 65 healthy controls were investigated in this study. Blood concentration of arginine was analyzed by automatic amino acid analyzer. The methylation of ASS gene promoter was evaluated by using methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing PCR (BSP) methods, and the mRNA level of ASS was evaluated by semi-quantitative RT-PCR. RESULTS: Blood concentration of arginine in PKU patients without dietary control was 0.017±0.009mmol/L while in normal persons was 0.129±0.007mmol/L, which is statistically significant (P<0.001). The promoter of ASS was methylated in PKU (15/15, 100%) but not in normal persons (0/15). The mRNA level of ASS in PKU patients was lower than that of normal people, which was well correlated with its methylation status. CONCLUSIONS: The silencing of ASS due to aberrant promoter CpG methylation may be an important mechanism for arginine biosynthesis disorders in PKU. High levels of phenylalanine and low levels of arginine are common characteristics in PKU patients. These findings would extend the current understanding of arginine, ASS in the development of PKU disease.