IKKα and IKKβ Regulation of DNA Damage-Induced Cleavage of Huntingtin

Background

Proteolysis of huntingtin (Htt) plays a key role in the pathogenesis of Huntington''s disease (HD). However, the environmental cues and signaling pathways that regulate Htt proteolysis are poorly understood. One stimulus may be the DNA damage that accumulates in neurons over time, and the subsequent activation of signaling pathways such as those regulated by IκB kinase (IKK), which can influence neurodegeneration in HD.

Methodology/Principal Findings

We asked whether DNA damage induces the proteolysis of Htt and if activation of IKK plays a role. We report that treatment of neurons with the DNA damaging agent etoposide or γ-irradiation promotes cleavage of wild type (WT) and mutant Htt, generating N-terminal fragments of 80–90 kDa. This event requires IKKβ and is suppressed by IKKα. Elevated levels of IKKα, or inhibition of IKKβ expression by a specific small hairpin RNA (shRNA) or its activity by sodium salicylate, prevents Htt proteolysis and increases neuronal resistance to DNA damage. Moreover, IKKβ phosphorylates the anti-apoptotic protein Bcl-xL, a modification known to reduce Bcl-xL levels, and activates caspases that can cleave Htt. When IKKβ expression is blocked, etoposide treatment does not decrease Bcl-xL and activation of caspases is diminished. Similar to silencing of IKKβ, increasing the level of Bcl-xL in neurons prevents etoposide-induced caspase activation and Htt proteolysis.

Conclusions/Significance

These results indicate that DNA damage triggers cleavage of Htt and identify IKKβ as a prominent regulator. Moreover, IKKβ-dependent reduction of Bcl-xL is important in this process. Thus, inhibition of IKKβ may promote neuronal survival in HD as well as other DNA damage-induced neurodegenerative disorders.     

Cell migration to CXCL12 requires simultaneous IKKα and IKKβ-dependent NF-κB signaling

CXCL12 and its unique receptor CXCR4, is critical for the homing of a variety of cell lineages during both development and tissue repair. CXCL12 is particularly important for the recruitment of hemato/lymphopoietic cells to their target organs. In conjunction with the damage-associated alarmin molecule HMGB1, CXCL12 mediates immune effector and stem/progenitor cell migration towards damaged tissues for subsequent repair. Previously, we showed that cell migration to HMGB1 simultaneously requires both IKKβ and IKKα-dependent NF-κB activation. IKKβ-mediated activation maintains sufficient expression of HMGB1's receptor RAGE, while IKKα-dependent NF-κB activation ensures continuous production of CXCL12, which complexes with HMGB1 to engage CXCR4. Here using fibroblasts and primary mature macrophages, we show that IKKβ and IKKα are simultaneously essential for cell migration in response to CXCL12 alone. Non-canonical NF-κB pathway subunits RelB and p52 are also both essential for cell migration towards CXCL12, suggesting that IKKα is required to drive non-canonical NF-κB signaling. Flow cytometric analyses of CXCR4 expression show that IKKβ, but not IKKα, is required to maintain a critical threshold level of this CXCL12 receptor. Time-lapse video microscopy experiments in primary MEFs reveal that IKKα is required both for polarization of cells towards a CXCL12 gradient and to establish a basal level of velocity towards CXCL12. In addition, CXCL12 modestly up-regulates IKKα-dependent p52 nuclear translocation and IKKα-dependent expression of the CXCL12 gene. On the basis of our collective results we posit that IKKα is needed to maintain the basal expression of a critical protein co-factor required for cell migration to CXCL12.     

NF-κB Activation Coordinated by IKKβ and IKKε Enables Latent Infection of Kaposi's Sarcoma-Associated Herpesvirus

All herpesviruses share a remarkable propensity to establish latent infection. Human Kaposi''s sarcoma-associated herpesvirus (KSHV) effectively enters latency after de novo infection, suggesting that KSHV has evolved with strategies to facilitate latent infection. NF-κB activation is imperative for latent infection of gammaherpesviruses. However, how NF-κB is activated during de novo herpesvirus infection is not fully understood. Here, we report that KSHV infection activates the inhibitor of κB kinase β (IKKβ) and the IKK-related kinase epsilon (IKKε) to enable host NF-κB activation and KSHV latent infection. Specifically, KSHV infection activated IKKβ and IKKε that were crucial for latent infection. Knockdown of IKKβ and IKKε caused aberrant lytic gene expression and impaired KSHV latent infection. Biochemical and genetic experiments identified RelA as a key player downstream of IKKβ and IKKε. Remarkably, IKKβ and IKKε were essential for phosphorylation of S⁵³⁶ and S⁴⁶⁸ of RelA, respectively. Phosphorylation of RelA S⁵³⁶ was required for phosphorylation of S⁴⁶⁸, which activated NF-κB and promoted KSHV latent infection. Expression of the phosphorylation-resistant RelA S⁵³⁶A increased KSHV lytic gene expression and impaired latent infection. Our findings uncover a scheme wherein NF-κB activation is coordinated by IKKβ and IKKε, which sequentially phosphorylate RelA in a site-specific manner to enable latent infection after KSHV de novo infection.     

TRAF2 Suppresses Basal IKK Activity in Resting Cells and TNFα Can Activate IKK in TRAF2 and TRAF5 Double Knockout Cells

Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF5 are adapter proteins involved in TNFα-induced activation of the c-Jun N-terminal kinase and nuclear factor κB (NF-κB) pathways. Currently, TNFα-induced NF-κB activation is believed to be impaired in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells. Here, we report instead that T2/5 DKO cells exhibit high basal IκB kinase (IKK) activity and elevated expression of NF-κB-dependent genes in unstimulated conditions. Although TNFα-induced receptor-interacting protein 1 ubiquitination is indeed impaired in T2/5 DKO cells, TNFα stimulation further increases IKK activity in these cells, resulting in significantly elevated expression of NF-κB target genes to a level higher than that in wild-type cells. Inhibition of NIK in T2/5 DKO cells attenuates basal IKK activity and restores robust TNFα-induced IKK activation to a level comparable with that seen in wild-type cells. This suggests that TNFα can activate IKK in the absence of TRAF2 and TRAF5 expression and receptor-interacting protein 1 ubiquitination. In addition, both the basal and TNFα-induced expression of anti-apoptotic proteins are normal in T2/5 DKO cells, yet these DKO cells remain sensitive to TNFα-induced cell death, due to the impaired recruitment of anti-apoptotic proteins to the TNFR1 complex in the absence of TRAF2. Thus, our data demonstrate that TRAF2 negatively regulates basal IKK activity in resting cells and inhibits TNFα-induced cell death by recruiting anti-apoptotic proteins to the TNFR1 complex rather than by activating the NF-κB pathway.     

Synthesis and biological evaluation of tricyclic anilinopyrimidines as IKKβ inhibitors

A series of tricyclic anilinopyrimidines were synthesized and evaluated as IKKβ inhibitors. Several analogues, including tricyclic phenyl (10, 18a, 18c, 18d, and 18j) and thienyl (26 and 28) derivatives were shown to have good in vitro enzyme potency and excellent cellular activity. Pharmaceutical profiling of a select group of tricyclic compounds compared to the non-tricyclic analogues suggested that in some cases, the improved cellular activity may be due to increased clog P and permeability.     

Phylogeny and evolutionary rates of G protein α subunit genes

Summary Rooted phylogenetic trees for a total of 34 genes encoding the stimulatory (s), inhibitory (i), transducin (t), G_x (x), G_z (z), G₁₁ (11), G₁₂ (12), G₁₃ (13), G₁₆ (16), G_q (q), and other (o) G protein a subunits have been constructed. The analysis shows that the G₁₂ (12 and 13), G_q (11, 16, and q), and G_s (s genes) groups form one cluster, and the G_x (x and z genes), G_i (i genes), G_t (t1 and t2), and G_o (o genes) groups form another cluster. During mammalian evolution, the rates of synonymous substitutions for these genes were estimated to be between 1.77 × 10^–9/site/year and 5.63 × 10^–9/site/year, whereas those of nonsynonymous substitutions were between 0.008 × 10^–9/site/year and 0.067 × 10^–9/site/year. These evolutionary rates are similar to those for histone genes, suggesting equally important biological functions of the G protein a subunits. Offprint requests to: S. Yokoyama     

Identification and expression of the medaka inhibin βE subunit

Activin E, a member of the TGF-β super family, is a protein dimer of mature inhibin βE subunits. Recently, it is reported that hepatic activin E may act as a hepatokine that alter whole body energy/glucose metabolism in human. However, orthologues of the activin E gene have yet to be identified in lower vertebrates, including fish. Here, we cloned the medaka (Oryzias latipes) activin E cDNA from liver. Among all the mammalian inhibin β subunits, the mature medaka activin E amino acid sequence shares the highest homology with mammalian activin E. Recombinant expression studies suggest that medaka activin E, the disulfide–bound mature form of mature inhibin βE subunits, may exert its effects in a way similar to that in mammals. Although activin E mRNA is predominantly expressed in liver in mammals, it is ubiquitously expressed in medaka tissues. Since expression in the liver was enhanced after a high fat diet, medaka activin E may be associated with energy/glucose metabolism, as shown in mice and human.

     

IKKα的研究进展

IκB激酶(IKK复合体)是NF-κB信号转导途径成员之一,包括3个亚基:催化亚基IKKα、IKKβ和调节亚基IKKγ。无刺激时,NF-κB与抑制蛋白IκB家族的一个成员结合,或者与无活性前体(如p100)结合而以无活性形式存在。在外界信号如TNF-α或淋巴毒素β等刺激下,经过复杂的信号转导,IKK复合体被激活,导致IκB和(或)p100发生磷酸化,结果NF-κB被释放出来,进入细胞核内激活靶基因。最新研究发现在TNF-α刺激下,IKKα可直接进入细胞核内,通过催化组蛋白H3磷酸化进而激活特定NF-κB应答基因的表达。IKKα是首次发现的信号转导途径中直接进入细胞核内调节基因表达的上游成分,为NF-κB信号转导途径的研究开辟了新的道路。     

The carboxyl-terminal helical domain of the ATP synthase γ subunit is involved in ε subunit conformation and energy coupling

The γ subunit located at the center of ATP synthase (F_OF₁) plays critical roles in catalysis. Escherichia coli mutant with Pro substitution of the γ subunit residue γLeu218, which are located the rotor shaft near the c subunit ring, decreased NADH-driven ATP synthesis activity and ATP hydrolysis-dependent H⁺ transport of membranes to ~60% and ~40% of the wild type, respectively, without affecting F_OF₁ assembly. Consistently, the mutant was defective in growth by oxidative phosphorylation, indicating that energy coupling is impaired by the mutation. The ε subunit conformations in the γLeu218Pro mutant enzyme were investigated by cross-linking between cysteine residues introduced into both the ε subunit (εCys118 and εCys134, in the second helix and the hook segment, respectively) and the γ subunit (γCys99 and γCys260, located in the globular domain and the carboxyl-terminal helix, respectively). In the presence of ADP, the two γ260 and ε134 cysteine residues formed a disulfide bond in both the γLeu218Pro mutant and the wild type, indicating that the hook segment of ε subunit penetrates into the α₃β₃-ring along with the γ subunits in both enzymes. However, γ260/ε134 cross-linking in the γLeu218Pro mutant decreased significantly in the presence of ATP, whereas this effect was small in the wild type. These results suggested that the γ subunit carboxyl-terminal helix containing γLeu218 is involved in the conformation of the ε subunit hook region during ATP hydrolysis and, therefore, is required for energy coupling in F_OF₁.     

Structural insights into IKKβ inhibition by natural products staurosporine and quercetin

This Letter describes the results of two combined approaches: homology modeling and molecular docking studies, in order to propose the molecular basis of IKKβ inhibition by staurosporine and quercetin as ATP-competitive inhibitors. The results provides a rationale and structural frameworks for designing potent ATP binding-site inhibitors of IKKβ, which is an attractive drug target for inflammatory diseases and has been found to be responsible for some of the already observed pharmacological effects for marketed drugs.     

Regulation of ATP hydrolysis by the ε subunit, ζ subunit and Mg-ADP in the ATP synthase of Paracoccus denitrificans

F₁F_O-ATP synthase is a crucial metabolic enzyme that uses the proton motive force from respiration to regenerate ATP. For maximum thermodynamic efficiency ATP synthesis should be fully reversible, but the enzyme from Paracoccus denitrificans catalyzes ATP hydrolysis at far lower rates than it catalyzes ATP synthesis, an effect often attributed to its unique ζ subunit. Recently, we showed that deleting ζ increases hydrolysis only marginally, indicating that other common inhibitory mechanisms such as inhibition by the C-terminal domain of the ε subunit (ε-CTD) or Mg-ADP may be more important. Here, we created mutants lacking the ε-CTD, and double mutants lacking both the ε-CTD and ζ subunit. No substantial activation of ATP hydrolysis was observed in any of these strains. Instead, hydrolysis in even the double mutant strains could only be activated by oxyanions, the detergent lauryldimethylamine oxide, or a proton motive force, which are all considered to release Mg-ADP inhibition. Our results establish that P. denitrificans ATP synthase is regulated by a combination of the ε and ζ subunits and Mg-ADP inhibition.     

Calcineurin B subunit interacts with proteasome subunit alpha type 7 and represses hypoxia-inducible factor-1α activity via the proteasome pathway

The calcineurin (CN) B subunit (CNB) is the regulatory subunit of CN, which is the only serine/threonine-specific protein phosphatase regulated by Ca²⁺/CaM. It has been shown to have potential as an anticancer agent, and has a positive effect on the phagocytic index and coefficient. We report here that CNB binds to proteasome subunit alpha type 7 (PSMA7) and inhibits the transactivation activity of hypoxia-inducible factor-1α (HIF-1α) via the proteasome pathway. In addition, we show that CNB represses the expression of vascular endothelial growth factor (VEGF), which is regulated by HIF-1α. These results indicate that CNB modulates cellular proteasome activity via a specific interaction with PSMA7. This may provide a molecular basis for its anticancer and antiviral activities.