REGγ Mitigates Radiation-Induced Enteritis by Preserving Mucin Secretion and Sustaining Microbiome Homeostasis.

Radiation-induced enteritis, a significant concern in abdominal radiation therapy, is associated closely with gut microbiota dysbiosis. The mucus layer plays a pivotal role in preventing the translocation of commensal and pathogenic microbes. Although significant expression of REGγ in intestinal epithelial cells is well established, its role in modulating the mucus layer and gut microbiota remains unknown. The current study revealed notable changes in gut microorganisms and metabolites in irradiated mice lacking REGγ, as compared to wild-type mice. Concomitant with gut microbiota dysbiosis, REGγ deficiency facilitated the infiltration of neutrophils and macrophages, thereby exacerbating intestinal inflammation after irradiation. Furthermore, fluorescence in situ hybridization assays unveiled an augmented proximity of bacteria to intestinal epithelial cells in REGγ knockout mice after irradiation. Mechanistically, deficiency of REGγ led to diminished goblet cell populations and reduced expression of key goblet cell markers, Muc2 and Tff3, observed in both murine models, minigut organoid systems and human intestinal goblet cells, indicating the intrinsic role of REGγ within goblet cells. Interestingly, although administration of broad-spectrum antibiotics did not alter the goblet cell numbers or mucin 2 (MUC2) secretion, it effectively attenuated inflammation levels in the ileum of irradiated REGγ absent mice, bringing them down to the wild-type levels. Collectively, these findings highlight the contribution of REGγ in counteracting radiation-triggered microbial imbalances and cell-autonomous regulation of mucin secretion.

Quercetin Mitigates Radiation-Induced Intestinal Injury and Promotes Intestinal Regeneration via Nrf2-Mediated Antioxidant Pathway1.

Radiation-induced intestinal injury is one the most common adverse events of radiotherapy, which can severely affect quality of life. There are currently no effective preventive and therapeutic options for this disorder. Quercetin is a natural flavonoid found in common food species, with the characteristics of antioxidative, anti-inflammatory, and anti-cancerous activity. However, the role of quercetin on radiation-induced intestinal injury and the underlying mechanism remains poorly understood. In this study, we found quercetin treatment can improve the survival rate of mice after a single-dose (10 Gy) abdominal irradiation. Quercetin-pretreated mice significantly reduced radiation-induced DNA damage and intestinal epithelium cell apoptosis. In addition, quercetin also improved the proliferation activity of intestinal stem cells and promoted intestine epithelium repair after irradiation. Further studies demonstrated that quercetin treatment curtailed radiation-induced reactive oxygen species generation via regulating Nrf2 signaling in intestinal epithelium cells. Furthermore, treatment with Nrf2 inhibitor, could reverse the above effects. Altogether, quercetin can ameliorate radiation-induced intestine injury via regulating Nrf2 signaling, scavenging free radicals, and promoting intestinal epithelium repair.

Reciprocal REGγ-Nrf2 Regulation Promotes Long Period ROS Scavenging in Oxidative Stress-Induced Cell Aging.

Increased accumulation of reactive oxygen species (ROS) and decline of adaptive response of antioxidants to oxidative stimuli has been implicated in the aging process. Nuclear factor erythroid 2-related factor 2 (Nrf2) activation is a core event in attenuating oxidative stress-associated aging. The activity is modulated by a more complex regulatory network. In this study, we demonstrate the proteasome activator REGγ function as a new regulator of Nrf2 activity upon oxidative stress in cell aging model induced by hydrogen peroxide (H2O2). REGγ deficiency promotes cell senescence in primary MEF cells after H2O2 treatment. Accordingly, ROS scavenging is accelerated in WT cells but blunted in REGγ lacking cells during 12-hour recovery from a 1-hour H2O2 treatment, indicating long-lasting antioxidant buffering capacity of REGγ. Mechanistically, through GSK-3ß inhibition, REGγ enhances the nuclear distribution and transcriptional activity of Nrf2, which is surveyed by induction of phase II enzymes including Ho1 and Nqo1. Meanwhile, Nrf2 mediates the transcriptional activation of REGγ upon H2O2 stimulation. More interestingly, short-term exposure to H2O2 leads to transiently upregulation and gradually descent of REGγ transcription, however sustained higher REGγ protein level even in the absence of H2O2 for 24 hours. Thus, our results establish a positive feedback loop between REGγ and Nrf2 and a new layer of adaptive response after oxidative stimulation that is the REGγ-GSK-3ß-Nrf2 pathway.

Atf7ip and Setdb1 interaction orchestrates the hematopoietic stem and progenitor cell state with diverse lineage differentiation.

Hematopoietic stem and progenitor cells (HSPCs) are a heterogeneous group of cells with expansion, differentiation, and repopulation capacities. How HSPCs orchestrate the stemness state with diverse lineage differentiation at steady condition or acute stress remains largely unknown. Here, we show that zebrafish mutants that are deficient in an epigenetic regulator Atf7ip or Setdb1 methyltransferase undergo excessive myeloid differentiation with impaired HSPC expansion, manifesting a decline in T cells and erythroid lineage. We find that Atf7ip regulates hematopoiesis through Setdb1-mediated H3K9me3 modification and chromatin remodeling. During hematopoiesis, the interaction of Atf7ip and Setdb1 triggers H3K9me3 depositions in hematopoietic regulatory genes including cebpß and cdkn1a, preventing HSPCs from loss of expansion and premature differentiation into myeloid lineage. Concomitantly, loss of Atf7ip or Setdb1 derepresses retrotransposons that instigate the viral sensor Mda5/Rig-I like receptor (RLR) signaling, leading to stress-driven myelopoiesis and inflammation. We find that ATF7IP or SETDB1 depletion represses human leukemic cell growth and induces myeloid differentiation with retrotransposon-triggered inflammation. These findings establish that Atf7ip/Setdb1-mediated H3K9me3 deposition constitutes a genome-wide checkpoint that impedes the myeloid potential and maintains HSPC stemness for diverse blood cell production, providing unique insights into potential intervention in hematological malignancy.

REGγ drives Lgr5+ stem cells to potentiate radiation induced intestinal regeneration.

Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), a marker of intestinal stem cells (ISCs), is considered to play key roles in tissue homoeostasis and regeneration after acute radiation injury. However, the activation of Lgr5 by integrated signaling pathways upon radiation remains poorly understood. Here, we show that irradiation of mice with whole-body depletion or conditional ablation of REGγ in Lgr5+ stem cell impairs proliferation of intestinal crypts, delaying regeneration of intestine epithelial cells. Mechanistically, REGγ enhances transcriptional activation of Lgr5 via the potentiation of both Wnt and Hippo signal pathways. TEAD4 alone or cooperates with TCF4, a transcription factor mediating Wnt signaling, to enhance the expression of Lgr5. Silencing TEAD4 drastically attenuated ß-catenin/TCF4 dependent expression of Lgr5. Together, our study reveals how REGγ controls Lgr5 expression and expansion of Lgr5+ stem cells in the regeneration of intestinal epithelial cells. Thus, REGγ proteasome appears to be a potential therapeutic target for radiation-induced gastrointestinal disorders.

REGγ regulates circadian clock by modulating BMAL1 protein stability.

Endogenous clocks generate rhythms in gene expression, which facilitates the organisms to cope through periodic environmental variations in accordance with 24-h light/dark time. A core question that needs to be elucidated is how such rhythms proliferate throughout the cells and regulate the dynamic physiology. In this study, we demonstrate the role of REGγ as a new regulator of circadian clock in mice, primary MEF, and SY5Y cells. Assessment of circadian conduct reveals a difference in circadian period, wheel mode, and the ability to acclimate the external light stimulus between WT and KO littermates. Compared to WT mice, REGγ KO mice attain the phase delay behavior upon light shock at early night. During the variation of 12/12 h light/dark (LD) exposure, levels of Per1, Per2, Cry1, Clock, Bmal1, and Rorα circadian genes in suprachiasmatic nucleus are significantly higher in REGγ KO than in WT mice, concomitant with remarkable changes in BMAL1 and PER2 proteins. In cultured cells depleted of REGγ, serum shock induces early response of the circadian genes Per1 and Per2 with the cyclic rhythm maintained. Mechanistic study indicates that REGγ directly degrades BMAL1 by the non-canonical proteasome pathway independent of ATP and ubiquitin. Silencing BMAL1 abrogates the changes in circadian genes in REGγ-deficient cells. However, inhibition of GSK-3ß, a known promoter for degradation of BMAL1, exacerbates the action of REGγ depletion. In conclusion, our findings define REGγ as a new factor, which functions as a rheostat of circadian rhythms to mitigate the levels of Per1 and Per2 via proteasome-dependent degradation of BMAL1.

Procyanidin B2 Promotes Intestinal Injury Repair and Attenuates Colitis-Associated Tumorigenesis via Suppression of Oxidative Stress in Mice.

Aims: Intact intestinal epithelium is essential to maintain normal intestinal physiological function. Irradiation-induced gastrointestinal syndrome or inflammatory bowel disease occurred when epithelial integrity was impaired. This study aims at exploring the mechanism of procyanidin B2 (PB2) administration to promote intestinal injury repair in mice. Results: PB2 treatment reduces reactive oxygen species (ROS) accumulation and protects the intestine damage from irradiation. Mechanistic studies reveal that PB2 could effectively slow down the degradation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and it significantly triggers Nrf2 into the nucleus, which leads to subsequent antioxidant enzyme expression. However, knockdown of Nrf2 attenuates PB2-induced protection in the intestine. More importantly, PB2 also promotes leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)-positive intestinal stem cells (Lgr5+ ISCs) driven regeneration via enhancing Wnt/ß-catenin signaling, which depends on, at least in part, activation of the Nrf2 signal. Evidence from an injury model of intestinal organoids is similar with in vivo results. Correspondingly, results from flow cytometric analysis and luciferase reporter assay reveal that PB2 also inhibits the level of ROS and promotes Lgr5 expression in vitro. Finally, PB2 alleviates the severity of experimental colitis and colitis-associated cancer in a long-term inflammatory model via inhibiting nuclear localization of p65. Innovation: This study, for the first time, reveals a role of PB2 for intestinal regeneration and repair after radiation or dextran sulfate sodium-induced injury in mice. Conclusion: Our results indicate that PB2 can repress oxidative stress via Nrf2/ARE signaling and then promote intestinal injury repair.

Reciprocal REGγ-mTORC1 regulation promotes glycolytic metabolism in hepatocellular carcinoma.

Despite significant progression in the study of hepatocellular carcinoma (HCC), the role of the proteasome in regulating cross talk between mTOR signaling and glycolysis in liver cancer progression is not fully understood. Here, we demonstrate that deficiency of REGγ, a proteasome activator, in mice significantly attenuates DEN-induced liver tumor formation. Ablation of REGγ increases the stability of PP2Ac (protein phosphatase 2 catalytic subunit) in vitro and in vivo, which dephosphorylates PRAS40 (AKT1 substrate 1) and stabilizes the interaction between PRAS40 and Raptor to inactive mTORC1-mediated hyper-glycolytic metabolism. In the DEN-induced animal model and clinical hepato-carcinoma samples, high levels of REGγ in HCC tumor regions contribute to reduced expression of PP2Ac, leading to accumulation of phosphorylated PRAS40 and mTORC1-mediated activation of HIF1α. Interestingly, mTORC1 enhances REGγ activity in HCC, forming a positive feedback regulatory loop. In conclusion, our study identifies REGγ-PP2Ac-PRAS40 axis as a new layer in regulating mTORC1 activity and downstream glycolytic alterations during HCC development, highlighting the REGγ-proteasome as a potential target for personalized HCC therapy.

Author Correction: The REGγ inhibitor NIP30 increases sensitivity to chemotherapy in p53-deficient tumor cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The REGγ inhibitor NIP30 increases sensitivity to chemotherapy in p53-deficient tumor cells.

A major challenge in chemotherapy is chemotherapy resistance in cells lacking p53. Here we demonstrate that NIP30, an inhibitor of the oncogenic REGγ-proteasome, attenuates cancer cell growth and sensitizes p53-compromised cells to chemotherapeutic agents. NIP30 acts by binding to REGγ via an evolutionarily-conserved serine-rich domain with 4-serine phosphorylation. We find the cyclin-dependent phosphatase CDC25A is a key regulator for NIP30 phosphorylation and modulation of REGγ activity during the cell cycle or after DNA damage. We validate CDC25A-NIP30-REGγ mediated regulation of the REGγ target protein p21 in vivo using p53-/- and p53/REGγ double-deficient mice. Moreover, Phosphor-NIP30 mimetics significantly increase the growth inhibitory effect of chemotherapeutic agents in vitro and in vivo. Given that NIP30 is frequently mutated in the TCGA cancer database, our results provide insight into the regulatory pathway controlling the REGγ-proteasome in carcinogenesis and offer a novel approach to drug-resistant cancer therapy.

REGγ controls Th17 cell differentiation and autoimmune inflammation by regulating dendritic cells.

Interleukin-17A (IL-17A)-producing helper T (Th17) cells are a subset of CD4+ T cells that play important pathological roles in autoimmune diseases. Although the intrinsic pathways of Th17 cell differentiation have been well described, how instructive signals derived from the innate immune system trigger the Th17 response and inflammation remains poorly understood. Here, we report that mice deficient in REGγ, a proteasome activator belonging to the 11S family, exhibit significantly deteriorated autoimmune neuroinflammation in an experimental autoimmune encephalomyelitis (EAE) model with augmented Th17 cell polarization in vivo. The results of the adoptive transfer of CD4+ T cells or dendritic cells (DCs) suggest that this phenotype is driven by DCs rather than T cells. Furthermore, REGγ deficiency promotes the expression of integrin αvß8 on DCs, which activates the maturation of TGF-ß1 to enhance Th17 cell development. Mechanistically, this process is mediated by the REGγ-proteasome-dependent degradation of IRF8, a transcription factor for αvß8. Collectively, our findings delineate a previously unknown mechanism by which REGγ-mediated protein degradation in DCs controls the differentiation of Th17 cells and the onset of an experimental autoimmune disease.

The REGγ-Proteasome Regulates Spermatogenesis Partially by P53-PLZF Signaling.

Development of spermatogonia and spermatocytes are the critical steps of spermatogenesis, impacting on male fertility. Investigation of the related regulators benefits the understanding of male reproduction. The proteasome system has been reported to regulate spermatogenesis, but the mechanisms and key contributing factors in vivo are poorly explored. Here we found that ablation of REGγ, a proteasome activator, resulted in male subfertility. Analysis of the mouse testes after birth showed there was a decreased number of PLZF+ spermatogonia and spermatocytes. Molecular analysis found that REGγ loss significantly increased the abundance of p53 protein in the testis, and directly repressed PLZF transcription in cell lines. Of note, allelic p53 haplodeficiency partially rescued the defects in spermatogenesis observed in REGγ-deficient mice. In summary, our results identify REGγ-p53-PLZF to be a critical pathway that regulates spermatogenesis and establishes a new molecular link between the proteasome system and male reproduction.

The proteasome activator REGγ counteracts immunoproteasome expression and autoimmunity.

For quite a long time, the 11S proteasome activator REGɑ and REGß, but not REGγ, are known to control immunoproteasome and promote antigen processing. Here, we demonstrate that REGγ functions as an inhibitor for immunoproteasome and autoimmune disease. Depletion of REGγ promotes MHC class I-restricted presentation to prime CD8+ T cells in vitro and in vivo. Mice deficient for REGγ have elevation of CD8+ T cells and DCs, and develop age-related spontaneous autoimmune symptoms. Mechanistically, REGγ specifically interacts with phosphorylated STAT3 and promotes its degradation in vitro and in cells. Inhibition of STAT3 dramatically attenuates levels of LMP2/LMP7 and antigen presentation in cells lacking REGγ. Importantly, treatment with STAT3 or LMP2/7 inhibitor prevented accumulation of immune complex in REGγ-/- kidney. Moreover, REGγ-/- mice also expedites Pristane-induced lupus. Bioinformatics and immunohistological analyses of clinical samples have correlated lower expression of REGγ with enhanced expression of phosphorylated STAT3, LMP2 and LMP7 in human Lupus Nephritis. Collectively, our results support the concept that REGγ is a new regulator of immunoproteasome to balance autoimmunity.

REGγ Contributes to Regulation of Hemoglobin and Hemoglobin δ Subunit.

Hemoglobin (Hb) is a family of proteins in red blood cells responsible for oxygen transport and vulnerable for oxidative damage. Hemoglobin δ subunit (HBD), a member of Hb family, is normally expressed by cells of erythroid lineage. Expression of Hb genes has been previously reported in nonerythroid and hematopoietic stem cells. Here, we report that Hb and HBD can be degraded via REGγ proteasome in hemopoietic tissues and nonerythroid cells. For this purpose, bone marrow, liver, and spleen hemopoietic tissues from REGγ+/+ and REGγ-/- mice and stable REGγ knockdown cells were evaluated for the degradation of Hb and HBD via REGγ. Western blot and immunohistochemical analyses exhibited downregulation of Hb in REGγ wild-type mouse tissues. This was validated by dynamic analysis following blockade of de novo synthesis of proteins with CHX. Degradation of HBD only occurred in REGγ WT cells but not in REGγN151Y, a dominant-negative REGγ mutant cell. Notably, downregulation of HBD was found in HeLa shN cells with stimulation of phenylhydrazine, an oxidation inducer, suggesting that the REGγ proteasome may target oxidatively damaged Hbs. In conclusion, our findings provide important implications for the degradation of Hb and HBD in hemopoietic tissues and nonerythroid cells via the REGγ proteasome.

Mutant p53 promotes cell spreading and migration via ARHGAP44.

The tumor suppressor p53 protein is either lost or mutated in about half of all human cancers. Loss of p53 function is well known to influence cell spreading, migration and invasion. While expression of mutant p53 is not equivalent to p53 loss, mutant p53 can acquire new functions to drive cell spreading and migration via different mechanisms. In our study, we found that mutant p53 significantly increased cell spreading and migration when comparing with p53-null cells. RNA-Seq analysis suggested that Rho GTPase activating protein 44 (ARHGAP44) is a new target of mutant p53, which suppressed ARHGAP44 transcription. ARHGAP44 has GAP activity and catalyze GTP hydrolysis on Cdc42. Higher level of GTP-Cdc42 was correlated with increase expression of mutant p53 and reduced ARHGAP44. Importantly, wt-ARHGAP44 but not mutant ARHGAP44 (R291A) suppressed mutant p53 mediated cell spreading and migration. Bioinformatics analysis indicated lower expression of ARHGAP44 in lung carcinoma compared with normal tissues, which was verified by RT-qPCR using specimens from patients. More interestingly, ARHGAP44 mRNA level was lower in tumors with mutant p53 than those with normal p53. Collectively, our results disclose a new mechanism by which mutant p53 stimulates cell spreading and migration.

IQGAP3 is essential for cell proliferation and motility during zebrafish embryonic development.

IQGAPs are scaffolding proteins that regulate actin assembly, exocyst function, cell motility, morphogenesis, adhesion and division. Vertebrates express 3 family members: IQGAP1, IQGAP2, and IQGAP3. IQGAP1 is known to stimulate nucleation of branched actin filaments through N-WASP and the Arp2/3 complex following direct binding to cytoplasmic tails of ligand-activated growth factor receptors, including EGFR, VEGFR2 and FGFR1. By contrast, little is known about functions of IQGAP2 or IQGAP3. Using in situ hybridization on whole mount zebrafish (Danio rerio) embryos, we show that IQGAP1 and IQGAP2 are associated with discrete tissues and organs, while IQGAP3 is mainly expressed in proliferative cells throughout embryonic and larval development. Morpholino knockdowns of IQGAP1 and IQGAP2 have little effect on embryo morphology while loss of function of IQGAP3 affects both cell proliferation and cell motility. IQGAP3 morphant phenotypes are similar to those resulting from overexpression of dominant negative forms of Ras or of Fibroblast Growth Factor Receptor 1 (FGFR1), suggesting that IQGAP3 plays a role in FGFR1-Ras-ERK signaling. In support of this hypothesis, dominant negative forms of FGFR1 or Ras could be rescued by co-injection of zebrafish IQGAP3 mRNA, strongly suggesting that IQGAP3 acts as a downstream regulator of the FGFR1-Ras signaling pathway.

Differential regulation of the REGγ-proteasome pathway by p53/TGF-ß signalling and mutant p53 in cancer cells.

Proteasome activity is frequently enhanced in cancer to accelerate metastasis and tumorigenesis. REGγ, a proteasome activator known to promote p53/p21/p16 degradation, is often overexpressed in cancer cells. Here we show that p53/TGF-ß signalling inhibits the REGγ-20S proteasome pathway by repressing REGγ expression. Smad3 and p53 interact on the REGγ promoter via the p53RE/SBE region. Conversely, mutant p53 binds to the REGγ promoter and recruits p300. Importantly, mutant p53 prevents Smad3/N-CoR complex formation on the REGγ promoter, which enhances the activity of the REGγ-20S proteasome pathway and contributes to mutant p53 gain of function. Depletion of REGγ alters the cellular response to p53/TGF-ß signalling in drug resistance, proliferation, cell cycle progression and proteasome activity. Moreover, p53 mutations show a positive correlation with REGγ expression in cancer samples. These findings suggest that targeting REGγ-20S proteasome for cancer therapy may be applicable to human tumours with abnormal p53/Smad protein status. Furthermore, this study demonstrates a link between p53/TGF-ß signalling and the REGγ-20S proteasome pathway, and provides insight into the REGγ/p53 feedback loop.

IFIT5 potentiates anti-viral response through enhancing innate immune signaling pathways.

Humans have a distinct combination of IFIT (IFN-induced protein with tetratricopeptide repeats) family orthologs, including IFIT1 (ISG56), IFIT2 (ISG54), IFIT3 (ISG60), and IFIT5 (ISG58). The function of IFIT1/IFIT2/IFIT3 has been intensively investigated. However, little is known about the role of IFIT5 in any cellular processes. In this study, we reported that both the mRNA and protein levels of IFIT5 are up-regulated in response to RNA virus infection or polyinosinic-cytidylic acid stimulation. Ectopic expression of IFIT5 could synergize IRF3- and NF-κB-mediated gene expression, whereas knockdown of IFIT5 impairs the transcription of these genes. Consistently, anti-viral responses of host cells are significantly increased or decreased in the presence or absence of IFIT5. Mechanistically, IFIT5 co-localizes partly with mitochondria and interacts with RIG-I and MAVS. Our study identified that IFIT5 is an important enhancer in innate immune response.

REGγ deficiency promotes premature aging via the casein kinase 1 pathway.

Our recent studies suggest a role for the proteasome activator REG (11S regulatory particles, 28-kDa proteasome activator)γ in the regulation of tumor protein 53 (p53). However, the molecular details and in vivo biological significance of REGγ-p53 interplay remain elusive. Here, we demonstrate that REGγ-deficient mice develop premature aging phenotypes that are associated with abnormal accumulation of casein kinase (CK) 1δ and p53. Antibody array analysis led us to identify CK1δ as a direct target of REGγ. Silencing CK1δ or inhibition of CK1δ activity prevented decay of murine double minute (Mdm)2. Interestingly, a massive increase of p53 in REGγ(-/-) tissues is associated with reduced Mdm2 protein levels despite that Mdm2 transcription is enhanced. Allelic p53 haplodeficiency in REGγ-deficient mice attenuated premature aging features. Furthermore, introducing exogenous Mdm2 to REGγ(-/-) MEFs significantly rescues the phenotype of cellular senescence, thereby establishing a REGγ-CK1-Mdm2-p53 regulatory pathway. Given the conflicting evidence regarding the "antiaging" and "proaging" effects of p53, our results indicate a key role for CK1δ-Mdm2-p53 regulation in the cellular aging process. These findings reveal a unique model that mimics acquired aging in mammals and indicates that modulating the activity of the REGγ-proteasome may be an approach for intervention in aging-associated disorders.

Site-specific acetylation of the proteasome activator REGγ directs its heptameric structure and functions.

The proteasome activator REGγ has been reported to promote degradation of steroid receptor coactivator-3 and cyclin-dependent kinase inhibitors p21, p16, and p19 in a ubiquitin- and ATP-independent manner. A recent comparative analysis of REGγ expression in mouse and human tissues reveals a unique pattern of REGγ in specific cell types, suggesting undisclosed functions and biological importance of this molecule. Despite the emerging progress made in REGγ-related studies, how REGγ function is regulated remains to be explored. In this study, we report for the first time that REGγ can be acetylated mostly on its lysine 195 (Lys-195) residue by CREB binding protein (CBP), which can be reversed by sirtuin 1 (SIRT1) in mammalian cells. Site-directed mutagenesis abrogated acetylation at Lys-195 and significantly attenuated the capability of REGγ to degrade its target substrates, p21 and hepatitis C virus core protein. Mechanistically, acetylation at Lys-195 is important for the interactions between REGγ monomers and ultimately influences REGγ heptamerization. Biological analysis of cells containing REGγ-WT or REGγ-K195R mutant indicates an impact of acetylation on REGγ-mediated regulation of cell proliferation and cell cycle progression. These findings reveal a previously unknown mechanism in the regulation of REGγ assembly and activity, suggesting a potential venue for the intervention of the ubiquitin-independent REGγ proteasome activity.