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1.  Amorphous Alumina Nanowire Array Efficiently Delivers Ac-DEVD-CHO to Inhibit Apoptosis of Dendritic Cells 
To create an effective well-ordered delivery platform still remains a challenge. Herein we fabricate vertically aligned alumina nanowire arrays via atomic layer deposition templated by carbon nanotubes. Using these arrays, a caspase-3/7 inhibitor was delivered into DC 2.4 cells and blocked apoptosis, as confirmed by fluorescence microscopy.
doi:10.1039/c3cc48088g
PMCID: PMC3929101  PMID: 24336780
2.  A negative feedback loop mediated by the Bcl6–cullin 3 complex limits Tfh cell differentiation 
The Journal of Experimental Medicine  2014;211(6):1137-1151.
Bcl6 and E3 ligase cullin 3 complexes mediate negative feedback regulation during thymocyte development and T cell activation to restrain exaggerated Tfh responses.
Induction of Bcl6 (B cell lymphoma 6) is essential for T follicular helper (Tfh) cell differentiation of antigen-stimulated CD4+ T cells. Intriguingly, we found that Bcl6 was also highly and transiently expressed during the CD4+CD8+ (double positive [DP]) stage of T cell development, in association with the E3 ligase cullin 3 (Cul3), a novel binding partner of Bcl6 which ubiquitinates histone proteins. DP stage–specific deletion of the E3 ligase Cul3, or of Bcl6, induced the derepression of the Bcl6 target genes Batf (basic leucine zipper transcription factor, ATF-like) and Bcl6, in part through epigenetic modifications of CD4+ single-positive thymocytes. Although they maintained an apparently normal phenotype after emigration, they expressed increased amounts of Batf and Bcl6 at basal state and produced explosive and prolonged Tfh responses upon subsequent antigen encounter. Ablation of Cul3 in mature CD4+ splenocytes also resulted in dramatically exaggerated Tfh responses. Thus, although previous studies have emphasized the essential role of Bcl6 in inducing Tfh responses, our findings reveal that Bcl6–Cul3 complexes also provide essential negative feedback regulation during both thymocyte development and T cell activation to restrain excessive Tfh responses.
doi:10.1084/jem.20132267
PMCID: PMC4042651  PMID: 24863065
3.  Novel Cul3 binding proteins function to remodel E3 ligase complexes 
BMC Cell Biology  2014;15:28.
Background
Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as leucine-rich repeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a BTB-Cul3-Rbx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein.
Results
Tandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides. After the SEQUEST algorithm and CDART program were used for protein identification and domain prediction, we discovered a group of Cul3-bound proteins that contain either the LRR or WD40 domain (CLWs). Further biochemical analysis revealed that the LRR domain-containing CLWs could bind both Cul3 and BTB domain-containing proteins. The dual binding role for the LRR domain-containing CLWs results in causing the BTB-domain protein to become a substrate instead of an adaptor.
To further distinguish potential substrates from other components that are part of the BCR ubiquitin ligase complex, we altered the parameters in the SEQUEST algorithm to select for peptide fragments with a modified lysine residue. This method not only identifies the potential substrates of the BCR ubiquitin ligase complex, but it also pinpoints the lysine residue in which the post-translational modification occurs. Interestingly, none of the CLWs were identified by this method, supporting our hypothesis that CLWs were not potential substrates but rather additional components of the BCR ubiquitin ligase complex.
Conclusion
Our study identified a new set of Cul3-binding proteins known as CLWs via tandem affinity purification and LC/MS-MS analysis. Subsequently, our biochemical analysis revealed that some CLWs modify binding of BTB domain-containing proteins to the complex, causing degradation of the BTB domain-containing protein. As these CLWs were excluded from our list of substrates, we propose that CLWs serve as unique Cul3 binding proteins that provide an alternative regulatory mechanism for the complex.
doi:10.1186/1471-2121-15-28
PMCID: PMC4107866  PMID: 25011449
Cullin3; Tandem-affinity purification; BTB domain-containing protein; BCR ubiquitin ligase complex; Mass spectrometry; E3 ubiquitin ligase; Protein purification; Ubiquitin; Ubiquitin ligase
4.  BTB-Kelch Proteins and Ubiquitination of Kainate Receptors 
Kainate receptors (KAR) form a class of glutamate receptors that have been implicated in epilepsy, stroke, Alzheimer’s and neuropathic pain.1 KAR subtypes are known to be segregated to specific locations within neurons and play significant roles in synaptic transmission and plasticity.2 Increasing evidence suggests a the role for ubiqutination in regulating the number of synaptic neurotransmitter receptors.3–5 The ubiquitin pathway consists of activation (E1), conjugation (E2) and ligation (E3). Cullins form the largest family of E3 ligase complexes. We have recently shown that the BTB/Kelch domain proteins, actinfilin and mayven, bind both Cul3 and specific KAR subtypes (GluR6 and GluR5-2b) to target these KARs for ubiquitination and degradation.5 In this chapter we will review how these interactions occur, what they mean for the stability of KARs and their associated proteins and how, in turn, they may affect synaptic functions in the central nervous system.
doi:10.1007/978-1-4419-9557-5_10
PMCID: PMC3929045  PMID: 21713671
5.  B-Myb promotes S-phase independently of its sequence-specific DNA binding activity and interacts with polymerase delta-interacting protein 1 (Pdip1) 
Cell Cycle  2012;11(21):4047-4058.
B-Myb is a highly conserved member of the Myb transcription factor family, which plays an essential role in cell cycle progression by regulating the transcription of genes at the G2/M-phase boundary. The role of B-Myb in other parts of the cell cycle is less well-understood. By employing siRNA-mediated silencing of B-Myb expression, we found that B-Myb is required for efficient entry into S-phase. Surprisingly, a B-Myb mutant that lacks sequence-specific DNA-binding activity and is unable to activate transcription of B-Myb target genes is able to rescue the S-phase defect observed after B-Myb knockdown. Moreover, we have identified polymerase delta-interacting protein 1 (Pdip1), a BTB domain protein known to bind to the DNA replication and repair factor PCNA as a novel B-Myb interaction partner. We have shown that Pdip1 is able to interact with B-Myb and PCNA simultaneously. In addition, we found that a fraction of endogenous B-Myb can be co-precipitated via PCNA, suggesting that B-Myb might be involved in processes related to DNA replication or repair. Taken together, our work suggests a novel role for B-Myb in S-phase that appears to be independent of its sequence-specific DNA-binding activity and its ability to stimulate the expression of bona fide B-Myb target genes.
doi:10.4161/cc.22386
PMCID: PMC3507500  PMID: 23032261
B-Myb; cell cycle; PCNA; Pdip1/S-phase; DNA-binding
6.  The Cullin3 Ubiquitin Ligase Functions as a Nedd8-bound Heterodimer 
Molecular Biology of the Cell  2007;18(3):899-909.
Cullins are members of a family of scaffold proteins that assemble multisubunit ubiquitin ligase complexes to confer substrate specificity for the ubiquitination pathway. Cullin3 (Cul3) forms a catalytically inactive BTB-Cul3-Rbx1 (BCR) ubiquitin ligase, which becomes functional upon covalent attachment of the ubiquitin homologue neural-precursor-cell-expressed and developmentally down regulated 8 (Nedd8) near the C terminus of Cul3. Current models suggest that Nedd8 activates cullin complexes by providing a recognition site for a ubiquitin-conjugating enzyme. Based on the following evidence, we propose that Nedd8 activates the BCR ubiquitin ligase by mediating the dimerization of Cul3. First, Cul3 is found as a neddylated heterodimer bound to a BTB domain-containing protein in vivo. Second, the formation of a Cul3 heterodimer is mediated by a Nedd8 molecule, which covalently attaches itself to one Cul3 molecule and binds to the winged-helix B domain at the C terminus of the second Cul3 molecule. Third, complementation experiments revealed that coexpression of two distinct nonfunctional Cul3 mutants can rescue the ubiquitin ligase function of the BCR complex. Likewise, a substrate of the BCR complex binds heterodimeric Cul3, suggesting that the Cul3 complex is active as a dimer. These findings not only provide insight into the architecture of the active BCR complex but also suggest assembly as a regulatory mechanism for activation of all cullin-based ubiquitin ligases.
doi:10.1091/mbc.E06-06-0542
PMCID: PMC1805106  PMID: 17192413
7.  BTB-ZF factors recruit the E3 ligase cullin 3 to regulate lymphoid effector programs 
Nature  2012;491(7425):618-621.
The differentiation of several T and B cell effector programs in the immune system is directed by signature transcription factors that induce rapid epigenetic remodeling. We report that PLZF, the BTB-ZF transcription factor directing the innate-like effector program of NKT thymocytes 1,2 was prominently associated with cullin 3 (Cul3), an E3 ubiquitin ligase previously shown to use BTB domain-containing proteins as adaptors for substrate binding 3–7. PLZF transported Cul3 to the nucleus where the two proteins were associated within a chromatin modifying complex. Furthermore, PLZF expression resulted in selective changes of ubiquitination of multiple components of this complex. Cul3 was also found associated with another BTB-ZF transcription factor, Bcl6, which directs the B cell germinal center and the T follicular helper programs. Conditional deletion in mice demonstrated an essential role of Cul3 for the development of PLZF- and Bcl6-dependent lineages. We conclude that distinct lineage-specific BTB-ZF transcription factors recruit Cul3 to alter the ubiquitination pattern of their associated chromatin modifying complex. We propose that this novel function is essential to direct the differentiation of several T and B lymphocyte effector programs, and may also be involved in the oncogenic role of PLZF and Bcl6 in leukemias and lymphomas 8,9.
doi:10.1038/nature11548
PMCID: PMC3504649  PMID: 23086144
8.  The cyclin E regulator cullin 3 prevents mouse hepatic progenitor cells from becoming tumor-initiating cells 
The Journal of Clinical Investigation  2010;120(11):3820-3833.
Cyclin E is often overexpressed in cancer tissue, leading to genetic instability and aneuploidy. Cullin 3 (Cul3) is a component of the BTB-Cul3-Rbx1 (BCR) ubiquitin ligase that is involved in the turnover of cyclin E. Here we show that liver-specific ablation of Cul3 in mice results in the persistence and massive expansion of hepatic progenitor cells. Upon induction of differentiation, Cul3-deficient progenitor cells underwent substantial DNA damage in vivo and in vitro, thereby triggering the activation of a cellular senescence response that selectively blocked the expansion of the differentiated offspring. Positive selection of undifferentiated progenitor cells required the expression of the tumor suppressor protein p53. Simultaneous loss of Cul3 and p53 in hepatic progenitors turned these cells into highly malignant tumor-initiating cells that formed largely undifferentiated tumors in nude mice. In addition, loss of Cul3 and p53 led to the formation of primary hepatocellular carcinomas. Importantly, loss of Cul3 expression was also detected in a large series of human liver cancers and correlated directly with tumor de-differentiation. The expression of Cul3 during hepatic differentiation therefore safeguards against the formation of progenitor cells that carry a great potential for transformation into tumor-initiating cells.
doi:10.1172/JCI41959
PMCID: PMC2964969  PMID: 20978349
9.  Pcif1 modulates Pdx1 protein stability and pancreatic β cell function and survival in mice 
The Journal of Clinical Investigation  2010;120(10):3713-3721.
The homeodomain transcription factor pancreatic duodenal homeobox 1 (Pdx1) is a major mediator of insulin transcription and a key regulator of the β cell phenotype. Heterozygous mutations in PDX1 are associated with the development of diabetes in humans. Understanding how Pdx1 expression levels are controlled is therefore of intense interest in the study and treatment of diabetes. Pdx1 C terminus–interacting factor-1 (Pcif1, also known as SPOP) is a nuclear protein that inhibits Pdx1 transactivation. Here, we show that Pcif1 targets Pdx1 for ubiquitination and proteasomal degradation. Silencing of Pcif1 increased Pdx1 protein levels in cultured mouse β cells, and Pcif1 heterozygosity normalized Pdx1 protein levels in Pdx1+/– mouse islets, thereby increasing expression of key Pdx1 transcriptional targets. Remarkably, Pcif1 heterozygosity improved glucose homeostasis and β cell function and normalized β cell mass in Pdx1+/– mice by modulating β cell survival. These findings indicate that in adult mouse β cells, Pcif1 limits Pdx1 protein accumulation and thus the expression of insulin and other gene targets important in the maintenance of β cell mass and function. They also provide evidence that targeting the turnover of a pancreatic transcription factor in vivo can improve glucose homeostasis.
doi:10.1172/JCI40440
PMCID: PMC2947215  PMID: 20811152
10.  The Ubiquitin Conjugating Enzyme, UbcM2, Engages in Novel Interactions with Components of Cullin-3 Based E3 Ligases† 
Biochemistry  2009;48(15):3527-3537.
The class III ubiquitin conjugating enzymes (E2s) are distinguished from other E2s by the presence of unique N-terminal domains, and the utilization of importin-11 for transport into the nucleus in an activation dependent fashion. To begin determining the physiological roles of these enzymes, we carried out a yeast two-hybrid screen with the class III E2, UbcM2. This screen retrieved RCBTB1, a putative substrate adaptor for a cullin3 (CUL3) E3 ligase. We initially established through biochemical studies that RCBTB1 has the properties of a CUL3 substrate adaptor. Further analysis of the UbcM2-RCBTB1 complex led to the discovery and characterization of the following novel interactions: (i) UbcM2 binds an N-terminal domain of CUL3 requiring the first 57 amino acids, the same domain that binds to RCBTB1 and other substrate adaptors; (ii) UbcM2 does not bind mutants of CUL3 that are deficient in substrate adaptor recruitment; (iii) UbcM2 interacts with CUL3 independent of a bridging RING-finger protein; and (iv) can engage the neddylated (i.e., activated) form of CUL3. We also present evidence that UbcM2 can bind to the N-terminal halves of multiple cullins, implying that this E2 is a general cofactor for this class of ligases. Together, these studies represent the first evidence that UbcM2, in concert with substrate adaptors, engages activated CUL3 ligases, thus suggesting that class III E2s are novel regulators of cullin ligases.
doi:10.1021/bi801971m
PMCID: PMC2680606  PMID: 19256485
11.  Constitutive Turnover of Cyclin E by Cul3 Maintains Quiescence▿ †  
Molecular and Cellular Biology  2007;27(10):3651-3666.
Two distinct pathways for the degradation of mammalian cyclin E have previously been described. One pathway is induced by cyclin E phosphorylation and is dependent on the Cul1/Fbw7-based E3 ligase. The other pathway is dependent on the Cul3-based E3 ligase, but the mechanistic details of this pathway have yet to be elucidated. To establish the role of Cul3 in the degradation of cyclin E in vivo, we created a conditional knockout of the Cul3 gene in mice. Interestingly, the biallelic loss of Cul3 in primary fibroblasts derived from these mice results in increased cyclin E expression and reduced cell viability, paralleling the loss of Cul3 protein expression. Cell cycle analysis of viable, Cul3 hypomorphic cells shows that decreasing the levels of Cul3 increases both cyclin E protein levels and the number of cells in S phase. In order to examine the role of Cul3 in an in vivo setting, we determined the effect of deletion of the Cul3 gene in liver. This gene deletion resulted in a dramatic increase in cyclin E levels as well as an increase in cell size and ploidy. The results we report here show that the constitutive degradation pathway for cyclin E that is regulated by the Cul3-based E3 ligase is essential to maintain quiescence in mammalian cells.
doi:10.1128/MCB.00720-06
PMCID: PMC1899986  PMID: 17339333

Results 1-11 (11)