Humans ; Mechanistic Target of Rapamycin Complex 2/metabolism* ; Multiprotein Complexes/metabolism* ; Neural Stem Cells/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rapamycin-Insensitive Companion of mTOR Protein/metabolism* ; TOR Serine-Threonine Kinases/metabolism*

The BCCIP (BRCA2- and CDKN1A-interacting protein) is an important cofactor for BRCA2 in tumor suppression. Although the low expression of BCCIP is observed in multiple clinically diagnosed primary tumor tissues such as ovarian cancer, renal cell carcinoma and colorectal carcinoma, the mechanism of how BCCIP is regulated in cells is still unclear. The human INO80/YY1 chromatin remodeling complex composed of 15 subunits catalyzes ATP-dependent sliding of nucleosomes along DNA. Here, we first report that BCCIP is a novel target gene of the INO80/YY1 complex by presenting a series of experimental evidence. Gene expression studies combined with siRNA knockdown data locked candidate genes including BCCIP of the INO80/YY1 complex. Silencing or over-expressing the subunits of the INO80/YY1 complex regulates the expression level of BCCIP both in mRNA and proteins in cells. Also, the functions of INO80/YY1 complex in regulating the transactivation of BCCIP were confirmed by luciferase reporter assays. Chromatin immunoprecipitation (ChIP) experiments clarify the enrichment of INO80 and YY1 at +0.17 kb downstream of the BCCIP transcriptional start site. However, this enrichment is significantly inhibited by either knocking down INO80 or YY1, suggesting the existence of both INO80 and YY1 is required for recruiting the INO80/YY1 complex to BCCIP promoter region. Our findings strongly indicate that BCCIP is a potential target gene of the INO80/YY1 complex.
Calcium-Binding Proteins ; genetics ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; Chromatin Assembly and Disassembly ; physiology ; DNA Helicases ; genetics ; metabolism ; HeLa Cells ; Humans ; Multiprotein Complexes ; genetics ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; Promoter Regions, Genetic ; physiology ; Transcription, Genetic ; physiology ; YY1 Transcription Factor ; genetics ; metabolism

An F-box protein, beta-TrCP recognizes substrate proteins and destabilizes them through ubiquitin-dependent proteolysis. It regulates the stability of diverse proteins and functions as either a tumor suppressor or an oncogene. Although the regulation by beta-TrCP has been widely studied, the regulation of beta-TrCP itself is not well understood yet. In this study, we found that the level of beta-TrCP1 is downregulated by various protein kinase inhibitors in triple-negative breast cancer (TNBC) cells. A PI3K/mTOR inhibitor PI-103 reduced the level of beta-TrCP1 in a wide range of TNBC cells in a proteasome-dependent manner. Concomitantly, the levels of c-Myc and cyclin E were also downregulated by PI-103. PI-103 reduced the phosphorylation of beta-TrCP1 prior to its degradation. In addition, knockdown of beta-TrCP1 inhibited the proliferation of TNBC cells. We further identified that pharmacological inhibition of mTORC2 was sufficient to reduce the beta-TrCP1 and c-Myc levels. These results suggest that mTORC2 regulates the stability of beta-TrCP1 in TNBC cells and targeting beta-TrCP1 is a potential approach to treat human TNBC.
Cell Line, Tumor ; Cell Proliferation ; Cell Survival/drug effects ; Cyclin E/genetics/metabolism ; Dose-Response Relationship, Drug ; Female ; Furans/pharmacology ; Gene Knockdown Techniques ; Humans ; Models, Biological ; Multiprotein Complexes/antagonists & inhibitors ; Phosphatidylinositol 3-Kinases/*antagonists & inhibitors ; Phosphorylation/drug effects ; Protein Kinase Inhibitors/*pharmacology ; Proteolysis/drug effects ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Pyridines/pharmacology ; Pyrimidines/pharmacology ; TOR Serine-Threonine Kinases/*antagonists & inhibitors ; Triple Negative Breast Neoplasms/genetics/*metabolism ; beta-Transducin Repeat-Containing Proteins/genetics/*metabolism

OBJECTIVETo investigate the expression of CSN complex (COP9 signal some subunits) in the patients with acute promyelocytic leukemia (APL) and its significance in the ATRA-induced APL differentiation.

METHODSUsing the NB4 cells as a model, morphologic observation and myeloid differentiation marker CD11b detection were used to monitor ATRA-induced APL differentiation, the expression of CSN complex in cell differentiation was detected by Western blot and reverse transcription real time fluorescent quantitative PCR (RT-qPCR) method. RT-qPCR was also used to detect the relative expression level of COP9 signalosome subunits in the APL patients and remission after treatment.

RESULTSATRA could obviously enhance CD11b expression; the cell morphology showed obvious differentiation characteristics. During the differentiation, the expression of COP9 signalosome subunits was down-regulated by ATRA. Meanwhile, the CSN expression level in newly diagnosed APL patients was much higher than that in controls (non-leukemia) (P < 0.05). The level of CSN expression was obviously down-regulated when APL patients achieved complete remission.

CONCLUSIONThe high CSN expression level in APL patients can be down-regulated by ATRA. CSN complex may have a significant effect on the pathogenesis and therapy of APL.

COP9 Signalosome Complex ; Cell Differentiation ; Cell Line, Tumor ; Down-Regulation ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; Multiprotein Complexes ; metabolism ; Peptide Hydrolases ; metabolism ; Tretinoin ; pharmacology

The long-term storage of memory requires the finely tuned coordination of intracellular signaling with the transcriptional, translational and epigenetic regulations of gene expression. Among the epigenetic mechanisms, however, we know relatively little about the involvement of chromatin remodeling-dependent control of gene expression in cognitive brain functions, compared with our knowledge of other such mechanisms (for example, histone modifications and DNA methylation). A few recent studies have implicated the Brm/Brg-associated factor (BAF) chromatin-remodeling complex, a mammalian homolog of the yeast Swi/Snf complex, in neuronal structural/functional plasticity and memory formation. The BAF complex was previously known to have a critical role in neurodevelopment, but these recent findings indicate that it also contributes to both cognitive functions in the adult brain and human mental disorders characterized by intellectual disability. In this review, we provide a brief overview of the BAF complexes, introduce recent research findings that link their functions to memory formation, and speculate on the yet-unknown molecular mechanisms that may be relevant to these processes.
Actins/metabolism ; Animals ; *Chromatin Assembly and Disassembly ; Chromosomal Proteins, Non-Histone/metabolism ; DNA-Binding Proteins/metabolism ; Gene Expression Regulation ; Humans ; Learning ; *Memory ; Multiprotein Complexes/metabolism ; Neurons/*metabolism ; Protein Binding ; Signal Transduction ; Transcription Factors/*metabolism

OBJECTIVETo explore apoptosis of multiple myeloma (MM) cells and its mechanism by the combined inhibition of mTORC2 signaling pathway and heat shock protein 90.

METHODSThe effects of Rapamycin, 17-AAG and the combination on proliferation of MM cell lines U266 and KM3 were assessed using MTT at different time points (0, 8, 24, 48 hour). Cell apoptosis and cell cycle distribution were measured by flow cytometry. The specific proteins p-AKT (ser473), p-AKT (thr450), p-S6 (S235/236) and AKT were detected by Western blotting.

RESULTSRapamycin, 17- AAG and the combination suppressed the proliferation of MM cell lines U266 and KM3, especially the combination of Rapamycin and 17-AAG synergistically inhibited the proliferation (P<0.05); Rapamycin induced G1 arrest both at 24 and 48 hours, 17-AAG also induced G1 arrest, especially at 48 hours (P<0.01); Rapamycin, 17-AAG alone decreased the expression of AKT and induced MM cell apoptosis to some extent (P<0.01); Chronic rapamycin treatment inhibited mTORC2; Inhibition of both mTORC2 and chaper on pathways degraded AKT and induced MM cell apoptosis, which was significantly higher than that of any single agent (P<0.01).

CONCLUSIONInhibition of both mTORC2 and chaper on pathways decreased the expression of AKT to induce apoptosis of MM cells in vitro.

Apoptosis ; Benzoquinones ; pharmacology ; Cell Cycle ; Cell Division ; Cell Line, Tumor ; drug effects ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; Lactams, Macrocyclic ; pharmacology ; Mechanistic Target of Rapamycin Complex 2 ; Multiple Myeloma ; pathology ; Multiprotein Complexes ; antagonists & inhibitors ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; Sirolimus ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism

Binding Sites ; Histone-Lysine N-Methyltransferase ; chemistry ; metabolism ; Histones ; chemistry ; metabolism ; Humans ; Methylation ; Multiprotein Complexes ; chemistry ; metabolism ; Nuclear Proteins ; chemistry ; metabolism ; Protein Binding ; Protein Conformation ; Yeasts ; chemistry

The inflammasome is a group of multiprotein complexes in the cytoplasm, which can activate caspase-1 that mediates the maturation and release of IL-1β, IL-18, IL-33 and other pro-inflammatory cytokines.NALP1 (NACHT leucine-rich-repeat protein 1), also known as NLRP1, is the first one of the identified complex inflammasomes with definite ligands mainly involved in the activation of inflammasome assembly and the formation of apoptotic bodies. Moreover, it was also found that NLRP1 plays an important biological role in the development of acute leukemia, the bone marrow hematopoietic stem cell apoptosis and other blood diseases. This review briefly summarizes the structure, activation mechanism, regulation and the role of NLRP1 in the hematopoietic system.
Adaptor Proteins, Signal Transducing ; metabolism ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Cytokines ; Hematologic Diseases ; metabolism ; pathology ; Humans ; Inflammasomes ; Multiprotein Complexes

Mammalian target of rapamycin (mTOR) plays essential roles in cell proliferation, survival and metabolism by forming at least two functional distinct multi-protein complexes, mTORC1 and mTORC2. External growth signals can be received and interpreted by mTORC2 and further transduced to mTORC1. On the other hand, mTORC1 can sense inner-cellular physiological cues such as amino acids and energy states and can indirectly suppress mTORC2 activity in part through phosphorylation of its upstream adaptors, IRS-1 or Grb10, under insulin or IGF-1 stimulation conditions. To date, upstream signaling pathways governing mTORC1 activation have been studied extensively, while the mechanisms modulating mTORC2 activity remain largely elusive. We recently reported that Sin1, an essential mTORC2 subunit, was phosphorylated by either Akt or S6K in a cellular context-dependent manner. More importantly, phosphorylation of Sin1 at T86 and T398 led to a dissociation of Sin1 from the functional mTORC2 holo-enzyme, resulting in reduced Akt activity and sensitizing cells to various apoptotic challenges. Notably, an ovarian cancer patient-derived Sin1-R81T mutation abolished Sin1-T86 phosphorylation by disrupting the canonical S6K-phoshorylation motif, thereby bypassing Sin1-phosphorylation-mediated suppression of mTORC2 and leading to sustained Akt signaling to promote tumorigenesis. Our work therefore provided physiological and pathological evidence to reveal the biological significance of Sin1 phosphorylation-mediated suppression of the mTOR/Akt oncogenic signaling, and further suggested that misregulation of this process might contribute to Akt hyper-activation that is frequently observed in human cancers.
Adaptor Proteins, Signal Transducing ; metabolism ; Animals ; Humans ; Mechanistic Target of Rapamycin Complex 1 ; Mechanistic Target of Rapamycin Complex 2 ; Models, Biological ; Multiprotein Complexes ; metabolism ; Phosphorylation ; Phosphothreonine ; metabolism ; TOR Serine-Threonine Kinases ; metabolism

Autophagy is a conserved and programmed catabolic process that degrades damaged proteins and organelles. But the underlying mechanism and functions of autophagy in the ischemia-reperfusion (IR)-induced injury are unknown. In this study, we employed simulated IR of N2a cells as an in vitro model of IR injury to the neurons and monitored autophagic processes. It was found that the levels of Beclin-1 (a key molecule of autophay complex, Beclin-1/class III PI3K) and LC-3II (an autophagy marker) were remarkably increased with time during the process of ischemia and the process of reperfusion after 90 min of ischemia, while the protein kinases p70S6K and mTOR which are involved in autophagy regulation showed delayed inactivation after reperfusion. Administration of 3-methyladenine (3MA), an inhibitor of class III PI3K, abolished autophagy during reperfusion, while employment of rapamycin, an inhibitor of mTORC1 (normally inducing autophagy), surprisingly weakened the induction of autophagy during reperfusion. Analyses of mitochondria function by relative cell viability demonstrated that autophagy inhibition by 3-MA attenuated the decline of mitochondria function during reperfusion. Our data demonstrated that there were two distinct dynamic patterns of autophagy during IR-induced N2a injury, Beclin-1/class III PI3K complex-dependent and mTORC1-dependent. Inhibition of over-autophagy improved cell survival. These suggest that targeting autophagy therapy will be a novel strategy to control IR-induced neuronal damage.
Adenine ; analogs & derivatives ; pharmacology ; Animals ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Autophagy ; Beclin-1 ; Cell Line, Tumor ; Cell Survival ; Mechanistic Target of Rapamycin Complex 1 ; Mice ; Mitochondria ; metabolism ; Multiprotein Complexes ; antagonists & inhibitors ; metabolism ; Neurons ; drug effects ; metabolism ; Neuroprotective Agents ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Reperfusion Injury ; metabolism ; Sirolimus ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism