Ho Sup Yoon

Solution structure of the antiapoptotic protein bcl-2

The structures of two isoforms of Bcl-2 that differ by two amino acids have been determined by NMR spectroscopy. Because wild-type Bcl-2 behaved poorly in solution, the structures were determined by using Bcl-2/Bcl-xL chimeras in which part of the putative unstructured loop of Bcl-2 was replaced with a shortened loop from Bcl-xL. These chimeric proteins have a low pI compared with the wild-type protein and are soluble. The structures of the two Bcl-2 isoforms consist of 6 α-helices with a hydrophobic groove on the surface similar to that observed for the homologous protein, Bcl-xL. Comparison of the Bcl-2 structures to that of Bcl-xL shows that although the overall fold is the same, there are differences in the structural topology and electrostatic potential of the binding groove. Although the structures of the two isoforms of Bcl-2 are virtually identical, differences were observed in the ability of the proteins to bind to a 25-residue peptide from the proapoptotic Bad protein and a 16-residue peptide from the proapoptotic Bak protein. These results suggest that there are subtle differences in the hydrophobic binding groove in Bcl-2 that may translate into differences in antiapoptotic activity for the two isoforms.

FKBP Family Proteins: Immunophilins with Versatile Biological Functions

Immunophilins consist of a family of highly conserved proteins binding with immunosuppressive drugs such as FK506, rapamycin and cyclosporin A. FK506-binding protein (FKBP) is one of two major immunophilins and most of FKBP family members bind FK506 and show peptidylprolyl cis/trans isomerase (PPIase) activity. Small size FKBP family members contain only FK506-binding domain, while FKBPs with large molecular weights possess extra domains such as tetratricopeptide repeat domains, calmodulin binding and transmembrane motifs. FKBPs are involved in several biochemical processes including protein folding, receptor signaling, protein trafficking and transcription. FKBP family proteins play important functional roles in the T-cell activation, when complexed with their ligands. The roles of immunophilins in protein transportation and apoptosis through their molecular interactions with receptors or proteins have emerged recently. Moreover, therapeutic implications of immunophilin ligands in treating neurodegenerative disorders have been accumulating. FK506 and its derivatives with no immunosuppressive activities bind to the conserved active sites of the canonical FKBP members such as FKBP12, which shows PPIase activity. These immunophilin ligands show variable efficacy in animal models for Parkinson’s disease, dementia, and spinal cord injury, where the canonical immunophilins function as chaperones and are associate with the protein folding and modulation of oxidative stress. On the other hand, in the noncanonical FKBP members such as FKBP38, FK506-binding site is not conserved and shows neither PPIase activity nor affinity to FK506. Interestingly, the small molecule-mediated inhibition of the noncanonical member of FKBP family appears to cause neuronal protection and induce proliferation of neuronal stem cells in a rat focal cerebral ischemia model. Currently, the mechanisms of actions remain unclear. This review focuses on molecular characteristics of the canonical and noncanonical FKBP family members and the biological functions of their ligands in performing neuroprotective and neurotrophic activities.

Structure of a new nucleic-acid-binding motif in eukaryotic transcriptional elongation factor TFIIS.

TRANSCRIPTIONAL elongation involves dynamic interactions among RNA polymerase and single-stranded and double-stranded nucleic acids in the ternary complex1—4. In prokaryotes its regulation pro-vides an important mechanism of genetic control1. Analogous eukaryotic mechanisms are not well understood5, but may control expression of proto-oncogenes6,7 and viruses, including the human immunodeficiency virus HIV-1 (ref. 8). The highly conserved euk-aryotic transcriptional elongation factor TFIIS9 enables RNA polymerase II (RNAPII) to read though pause or termination sites, nucleosomes and sequence-specific DNA-binding proteins10—14. Two distinct domains of human TFIIS, which bind RNAPII and nucleic acids, regulate read-through10 and possibly nascent transcript cleavage11—15. Here we describe the three-dimensional NMR16 structure of a Cys4 nucleic-acid-binding domain from human TFIIS9—10. Unlike previously characterized zinc modules17—21, which contain an a-helix, this structure consists of a three-stranded β-sheet. Analogous Cys4 structural motifs may occur in other proteins involved in DNA or RNA trans-actions22—24, including RNAPII itself25. This new structure, desig-nated the Zn ribbon, extends the repertoire of Zn-mediated peptide architectures26 and highlights the growing recognition of the β-sheet as a motif of nucleic-acid recognition27—28.

Mitotic Histone H3 Phosphorylation by Vaccinia-Related Kinase 1 in Mammalian Cells†

ABSTRACT Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.

PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex

Endoplasmic reticulum (ER)–Golgi membrane transport and autophagy are intersecting trafficking pathways that are tightly regulated and crucial for homeostasis, development and disease. Here, we identify UVRAG, a beclin-1-binding autophagic factor, as a phosphatidylinositol-3-phosphate (PtdIns(3)P)-binding protein that depends on PtdIns(3)P for its ER localization. We further show that UVRAG interacts with RINT-1, and acts as an integral component of the RINT-1-containing ER tethering complex, which couples phosphoinositide metabolism to COPI-vesicle tethering. Displacement or knockdown of UVRAG profoundly disrupted COPI cargo transfer to the ER and Golgi integrity. Intriguingly, autophagy caused the dissociation of UVRAG from the ER tether, which in turn worked in concert with the Bif-1–beclin-1–PI(3)KC3 complex to mobilize Atg9 translocation for autophagosome formation. These findings identify a regulatory mechanism that coordinates Golgi–ER retrograde and autophagy-related vesicular trafficking events through physical and functional interactions between UVRAG, phosphoinositide and their regulatory factors, thereby ensuring spatiotemporal fidelity of membrane trafficking and maintenance of organelle homeostasis.

A Light-Driven Therapy of Pancreatic Adenocarcinoma Using Gold Nanorods-Based Nanocarriers for Co-Delivery of Doxorubicin and siRNA.

In this work, we report the engineering of polyelectrolyte polymers coated Gold nanorods (AuNRs)-based nanocarriers that are capable of co-delivering small interfering RNA (siRNA) and an anticancer drug doxorubicin (DOX) to Panc-1 cancer cells for combination of both chemo- and siRNA-mediated mutant K-Ras gene silencing therapy. Superior anticancer efficacy was observed through synergistic combination of promoted siRNA and DOX release upon irradiating the nanoplex formulation with 665 nm light. Our antitumor study shows that the synergistic effect of AuNRs nanoplex formulation with 665 nm light treatment is able to inhibit the in vivo tumor volume growth rate by 90%. The antitumor effect is contributed from the inactivation of K-Ras gene and thereby causing a profound synthesis (S) phase arrest in treated Panc-1 cells. Our study shows that the percentage of Panc-1 cells treated by nanoplex formulation with S phase is determined to be 35% and it is 17% much higher than that of Panc-1 cells without any treatments. The developed nanotherapy formulation here, that combines chemotherapy, RNA silencing and NIR window light-mediated therapy, will be seen to be the next natural step to be taken in the clinical research for improving the therapeutic outcomes of the pancreatic adenocarcinoma treatment.

FKBP38 Protects Bcl-2 from Caspase-dependent Degradation

The cellular processes that regulate Bcl-2 at the posttranslational levels are as important as those that regulate bcl-2 synthesis. Previously we demonstrated that the suppression of FK506-binding protein 38 (FKBP38) contributes to the instability of Bcl-2 or leaves Bcl-2 unprotected from degradation in an unknown mechanism. Here, we studied the underlying molecular mechanism mediating this process. We first showed that Bcl-2 binding-defective mutants of FKBP38 fail to accumulate Bcl-2 protein. We demonstrated that the FKBP38-mediated Bcl-2 stability is specific as the levels of other anti-apoptotic proteins such as Bcl-XL and Mcl-1 remained unaffected. FKBP38 enhanced the Bcl-2 stability under the blockade of de novo protein synthesis, indicating it is posttranslational. We showed that the overexpression of FKBP38 attenuates reduction rate of Bcl-2, thus resulting in an increment of the intracellular Bcl-2 level, contributing to the resistance of apoptotic cell death induced by the treatment of kinetin riboside, an anticancer drug. Caspase inhibitors markedly induced the accumulation of Bcl-2. In caspase-3-activated cells, the knockdown of endogenous FKBP38 by small interfering RNA resulted in Bcl-2 down-regulation as well, which was significantly recovered by the treatment with caspase inhibitors or overexpression of FKBP38. Finally we presented that the Bcl-2 cleavage by caspase-3 is blocked when Bcl-2 binds to FKBP38 through the flexible loop. Taken together, these results suggest that FKBP38 is a key player in regulating the function of Bcl-2 by antagonizing caspase-dependent degradation through the direct interaction with the flexible loop domain of Bcl-2, which contains the caspase cleavage site.

Molecular mimicry-based repositioning of nutlin-3 to anti-apoptotic Bcl-2 family proteins.

The identification of off-target binding of drugs is a key to repositioning drugs to new therapeutic categories. Here we show the universal interactions of the p53 transactivation domain (p53TAD) with various anti-apoptotic Bcl-2 family proteins via a mouse double minute 2 (MDM2) binding motif, which play an important role in transcription-independent apoptotic pathways of p53. Interestingly, our structural studies reveal that the anti-apoptotic Bcl-2 family proteins and MDM2 share a similar mode of interaction with the p53TAD. On the basis of this close molecular mimicry, our NMR results demonstrate that the potent MDM2 antagonists Nutlin-3 and PMI bind to the anti-apoptotic Bcl-2 family proteins in a manner analogous to that with the p53TAD.

Dual-site Interactions of p53 Protein Transactivation Domain with Anti-apoptotic Bcl-2 Family Proteins Reveal a Highly Convergent Mechanism of Divergent p53 Pathways

Background: Interactions between p53 and Bcl-2 family proteins serve a critical role in transcription-independent p53 apoptosis. Results: We studied the interactions of p53TAD2 with anti-apoptotic Bcl-2 family proteins at the atomic level by NMR, mutagenesis, and structure calculation. Conclusion: Bcl-XL/Bcl-2, MDM2, and CBP/p300 share similar modes of binding to the dual p53TAD motifs. Significance: Dual-site interaction of p53TAD is a highly conserved mechanism in the transcription-dependent and transcription-independent p53 apoptotic pathways. Molecular interactions between the tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins play an important role in the transcription-independent apoptosis of p53. The p53 transactivation domain (p53TAD) contains two conserved ΦXXΦΦ motifs (Φ indicates a bulky hydrophobic residue and X is any other residue) referred to as p53TAD1 (residues 15–29) and p53TAD2 (residues 39–57). We previously showed that p53TAD1 can act as a binding motif for anti-apoptotic Bcl-2 family proteins. In this study, we have identified p53TAD2 as a binding motif for anti-apoptotic Bcl-2 family proteins by using NMR spectroscopy, and we calculated the structures of Bcl-XL/Bcl-2 in complex with the p53TAD2 peptide. NMR chemical shift perturbation data showed that p53TAD2 peptide binds to diverse members of the anti-apoptotic Bcl-2 family independently of p53TAD1, and the binding between p53TAD2 and p53TAD1 to Bcl-XL is competitive. Refined structural models of the Bcl-XL·p53TAD2 and Bcl-2·p53TAD2 complexes showed that the binding sites occupied by p53TAD2 in Bcl-XL and Bcl-2 overlap well with those occupied by pro-apoptotic BH3 peptides. Taken together with the mutagenesis, isothermal titration calorimetry, and paramagnetic relaxation enhancement data, our structural comparisons provided the structural basis of p53TAD2-mediated interaction with the anti-apoptotic proteins, revealing that Bcl-XL/Bcl-2, MDM2, and cAMP-response element-binding protein-binding protein/p300 share highly similar modes of binding to the dual p53TAD motifs, p53TAD1 and p53TAD2. In conclusion, our results suggest that the dual-site interaction of p53TAD is a highly conserved mechanism underlying target protein binding in the transcription-dependent and transcription-independent apoptotic pathways of p53.

Macro Histone H2A1.2 (MacroH2A1) Protein Suppresses Mitotic Kinase VRK1 during Interphase

Background: VRK1 phosphorylates mitotic histone H3 at Thr-3 and Ser-10, but its negative regulator was not elucidated during interphase. Results: The macrodomain of macroH2A1 interacts with VRK1, and this suppresses enzymatic activity of VRK1 during interphase. Conclusion: Specific binding between VRK1 and macroH2A1 is required to regulate the cell cycle-dependent histone H3 phosphorylation. Significance: Understanding epigenetic regulation of histone H3 during the cell cycle is important in cancer development. VRK1-mediated phosphorylation of histone H3 should be restricted in mitosis for consistent cell cycling, and defects in this process trigger cellular catastrophe. However, an interphasic regulator against VRK1 has not been actually investigated so far. Here, we show that the histone variant macrodomain-containing histone H2A1.2 functions as a suppressor against VRK1 during interphase. The level of macroH2A1.2 was markedly reduced in the mitotic phase, and the macroH2A1.2-mediated inhibition of histone H3 phosphorylation occurred mainly during interphase. We also found direct interaction and binding features between VRK1 and macroH2A1.2 by NMR spectroscopy. Hence, our findings might provide valuable insight into the underlying molecular mechanism regarding an epigenetic regulation of histone H3 during the cell cycle.