Ebrahim Zandi

Bridging the Gap: Composition, Regulation, and Physiological Function of the IκB Kinase Complex

Protein kinases that regulate the activity of specific transcription factors in response to extracellular stimuli not only are the subject of intense research but also are being chased as potential targets for development of new drugs for treatment of various human diseases. One such protein kinase is IKK, the IκB kinase that activates nuclear factor κB (NF-κB) through phosphorylation of IκB inhibitory proteins. In this review, we summarize the discovery of IKK and recent knowledge about its composition, regulation, and physiological functions. NF-κB transcription factors regulate the expression of a large number of genes that are necessary for proper functioning of the immune system and are key mediators of inflammatory responses to pathogens (2, 4, 5). NF-κB is also associated with cellular transformation and oncogenesis, and one of its most important, but lately discovered functions, is the activation of an antiapoptotic gene expression program (6, 29, 36, 38, 39, 42). As a transcription factor that orchestrates the inflammatory response, NF-κB is rapidly activated, independently of new protein synthesis, in response to signals produced during infection (e.g., bacterial endotoxins and viral double-stranded RNA) (for a review, see reference 3). NF-κB activation is also a transient response; this is of importance because many of the genes that are activated by NF-κB encode potentially toxic products such as tumor necrosis factor (TNF). The key to NF-κB regulation is the inhibitory κB (IκB) proteins which retain NF-κB in the cytoplasm (reviewed in reference 37). In response to diverse stimuli, IκBs are rapidly degraded and the freed NF-κB dimers translocate to the nucleus. Several years ago, it was established that the critical event which triggers the polyubiquitination and degradation of IκBs via the 26S proteasome is their stimulus-dependent phosphorylation at two serine residues (residues 32 and 36 in IκBα) that are located within their conserved N-terminal regulatory region (1, 10–12, 18, 20, 34, 40). The protein kinase that phosphorylates these regulatory sites remained elusive, and without detailed knowledge about its molecular identity, there was little progress towards a full understanding of the signaling pathways that control NF-κB activity. The initial hunt for such a protein kinase yielded many false candidates, such as protein kinase C, casein kinase II, and ribosomal S6 kinase (pp90rsk) (reviewed in reference 43). Although most of these kinases phosphorylate IκB proteins in the test tube on different serine, threonine, or tyrosine residues, none of them was found to phosphorylate the two regulatory sites that trigger the degradation of IκBs in a stimulus-dependent manner. A large-molecular-mass (700-kDa) protein kinase activity that phosphorylates IκBα on S32 and S36 in a ubiquitin-dependent manner was also detected in extracts of nonstimulated HeLa cells (13, 25). However, this activity was not reported to be stimulus dependent, and to date, its components and molecular identity are unknown. A careful consideration of IκB phosphorylation indicated that the physiological IκB kinase had to fulfill several criteria. Its activity should be stimulated by inducers of NF-κB with kinetics that are consistent with those of NF-κB activation, and it should phosphorylate both S32 and S36 in the N terminus of IκBα and both S19 and S23 in the N terminus of IκBβ. In addition, since substitution of threonines for these serines results in IκB mutants that are resistant to degradation, the physiological IκB kinase should be serine specific (18).

Identification of post-translational modifications by blind search of mass spectra

Most tandem mass spectrometry (MS/MS) database search algorithms perform a restrictive search that takes into account only a few types of post-translational modifications (PTMs) and ignores all others. We describe an unrestrictive PTM search algorithm, MS-Alignment, that searches for all types of PTMs at once in a blind mode, that is, without knowing which PTMs exist in nature. Blind PTM identification makes it possible to study the extent and frequency of different types of PTMs, still an open problem in proteomics. Application of this approach to lens proteins resulted in the largest set of PTMs reported in human crystallins so far. Our analysis of various MS/MS data sets implies that the biological phenomenon of modification is much more widespread than previously thought. We also argue that MS-Alignment reveals some uncharacterized modifications that warrant further experimental validation.

Signaling Role of Intracellular Iron in NF-κB Activation

Iron chelators inhibit endotoxin-induced NF-κB activation in hepatic macrophages (HMs), suggesting a role for the intracellular chelatable pool of iron in NF-κB activation. The present study tested this hypothesis. Analysis of Fe59-loaded HMs stimulated with lipopolysaccharide (LPS), revealed a previously unreported, transient rise in intracellular low molecular weight (LMW)·Fe59complex ([LMW·Fe] i ) at ≤2 min returning to the basal level within 15 min. The [LMW·Fe] i response preceded IκB kinase (IKK) (≥15 min) and NF-κB (≥30 min) activation. Iron chelators (1,2-dimethyl-3-hydroxypyridin-4-one andN,N′-bis-2-hydroxybenzylethylenediamine-N,N′-diacetic acid) abrogated the [LMW·Fe] i response and IKK and NF-κB activation. The [LMW·Fe] i response was also observed in tumor necrosis factor α (TNFα)-stimulated HMs and RAW264.7 cells treated with LPS and interferon-γ but not in primary rat hepatocytes or myofibroblastic cells exposed to LPS or TNFα. Both [LMW·Fe] i response and IKK activation in LPS-stimulated HMs were inhibited by diphenylene iodonium (nonspecific inhibitor for flavin-containing oxidases),l-N 6-(1-iminoethyl)lysine (selective iNOS inhibitor), and adenoviral-mediated expression of a dominant negative mutant of Rac1 or Cu,Zn-superoxide dismutase, suggesting the role of ⋅NO and O 2 ⨪ in mediating the iron signaling. In fact, this inhibition was recapitulated by a cell-permeable scavenger of ONOO−, 5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinato iron (III) chloride. Conversely, ONOO− alone induced both [LMW·Fe] i response and IKK activation. Finally, direct addition of ferrous iron to cultured HMs activated IKK and NF-κB. These results support a novel signaling role for [LMW·Fe] i in IKK activation, which appears to be induced by ONOO−and selectively operative in macrophages.

Discovery of an orally active small-molecule irreversible inhibitor of protein disulfide isomerase for ovarian cancer treatment

Protein disulfide isomerase (PDI), an endoplasmic reticulum chaperone protein, catalyzes disulfide bond breakage, formation, and rearrangement. The effect of PDI inhibition on ovarian cancer progression is not yet clear, and there is a need for potent, selective, and safe small-molecule inhibitors of PDI. Here, we report a class of propynoic acid carbamoyl methyl amides (PACMAs) that are active against a panel of human ovarian cancer cell lines. Using fluorescent derivatives, 2D gel electrophoresis, and MS, we established that PACMA 31, one of the most active analogs, acts as an irreversible small-molecule inhibitor of PDI, forming a covalent bond with the active site cysteines of PDI. We also showed that PDI activity is essential for the survival and proliferation of human ovarian cancer cells. In vivo, PACMA 31 showed tumor targeting ability and significantly suppressed ovarian tumor growth without causing toxicity to normal tissues. These irreversible small-molecule PDI inhibitors represent an important approach for the development of targeted anticancer agents for ovarian cancer therapy, and they can also serve as useful probes for investigating the biology of PDI-implicated pathways.

DNA-PKcs dependence of Artemis endonucleolytic activity, differences between hairpins and 5' or 3' overhangs.

During V(D)J recombination, the RAG proteins create DNA hairpins at the V, D, or J coding ends, and the structure-specific nuclease Artemis is essential to open these hairpins prior to joining. Artemis also is an endonuclease for 5′ and 3′ overhangs at many DNA double strand breaks caused by ionizing radiation, and Artemis functions as part of the nonhomologous DNA end joining pathway in repairing these. All of these activities require activation of the Artemis protein by interaction with and phosphorylation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In this study, we have identified a region of the Artemis protein involved in the interaction with DNA-PKcs. Furthermore, the biochemical and functional analyses of C-terminally truncated Artemis variants indicate that the hair-pin opening and DNA overhang endonucleolytic features of Artemis are triggered by DNA-PKcs in two modes. First, autoinhibition mediated by the C-terminal tail of Artemis is relieved by phosphorylation of this tail by DNA-PKcs. Thus, C-terminally truncated Artemis derivatives imitate DNA-PKcs-activated wild type Artemis protein and exhibit intrinsic hairpin opening activity. Second, DNA-PKcs may optimally configure 5′ and 3′ overhang substrates for the endonucleolytic function of Artemis.

Hydrogen Sulfide Promotes Tet1- and Tet2-Mediated Foxp3 Demethylation to Drive Regulatory T Cell Differentiation and Maintain Immune Homeostasis

Regulatory T (Treg) cells are essential for maintenance of immune homeostasis. Here we found that hydrogen sulfide (H2S) was required for Foxp3(+) Treg cell differentiation and function and that H2S deficiency led to systemic autoimmune disease. H2S maintained expression of methylcytosine dioxygenases Tet1 and Tet2 by sulfhydrating nuclear transcription factor Y subunit beta (NFYB) to facilitate its binding to Tet1 and Tet2 promoters. Transforming growth factor-β (TGF-β)-activated Smad3 and interleukin-2 (IL-2)-activated Stat5 facilitated Tet1 and Tet2 binding to Foxp3. Tet1 and Tet2 catalyzed conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in Foxp3 to establish a Treg-cell-specific hypomethylation pattern and stable Foxp3 expression. Consequently, Tet1 and Tet2 deletion led to Foxp3 hypermethylation, impaired Treg cell differentiation and function, and autoimmune disease. Thus, H2S promotes Tet1 and Tet2 expression, which are recruited to Foxp3 by TGF-β and IL-2 signaling to maintain Foxp3 demethylation and Treg-cell-associated immune homeostasis.

Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation.

Phagocytosis and autophagy are two important and related arms of the hosts first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating its phagosomal trafficking to induce a burst of reactive oxygen species (ROS) and inflammatory cytokines. Consequently, ectopic expression or depletion of Rubicon profoundly affected ROS, inflammatory cytokine production, and subsequent antimicrobial activity. Rubicons actions in autophagy and in the NADPH oxidase complex are functionally and genetically separable, indicating that Rubicon functions in two ancient innate immune machineries, autophagy and phagocytosis, depending on the environmental stimulus. Rubicon may thus be pivotal to generating an optimal intracellular immune response against microbial infection.

Identification of Galectin-3-binding Protein as a Factor Secreted by Tumor Cells That Stimulates Interleukin-6 Expression in the Bone Marrow Stroma

We have previously demonstrated that neuroblastoma cells increase the expression of interleukin-6 by bone marrow stromal cells and that stimulation does not require cell-cell contact. In this study we report the purification and identification of a protein secreted by neuroblastoma cells that stimulates interleukin-6 production by stromal cells. Using a series of chromatographic purification steps including heparin-affinity, ion exchange, and molecular sieve chromatography followed by trypsin digestion and liquid chromatography tandem mass spectrometry, we identified in serum-free conditioned medium of neuroblastoma cells several secreted peptides including galectin-3-binding protein, also known as 90-kDa Mac-2-binding protein. We demonstrated the presence of the galectin-3-binding protein in the conditioned medium of several neuroblastoma cell lines and in chromatographic fractions with interleukin-6 stimulatory activity. Consistently, bone marrow stromal cells express galectin-3, the receptor for galectin-3-binding protein. Supporting a role for galectin-3-binding protein in stimulating interleukin-6 expression in bone marrow stromal cells, we observed that recombinant galectin-3-binding protein stimulated interleukin-6 expression in these cells and that interleukin-6 stimulation by neuroblastoma-conditioned medium was inhibited in the presence of lactose or a neutralizing anti-galectin-3 antibody. Down-regulation of galectin-3-binding protein expression in neuroblastoma cells also decreased the interleukin-6 stimulatory activity of the conditioned medium on bone marrow stromal cells. We also provide evidence that stimulation of interleukin-6 by galectin-3-binding protein involves activation of the Erk1/2 pathway. The data, thus, identifies galectin-3-binding protein as a factor secreted by neuroblastoma cells that stimulates the expression of interleukin-6 in bone marrow stromal cells and provides a novel function for this protein in cancer progression.

Regulation of IκB kinase (IKK) complex by IKKγ-dependent phosphorylation of the T-loop and C terminus of IKKβ

The mechanistic relationship of phosphorylation of the C terminus of IKKβ with phosphorylation of its T-loop kinase domain within the IKK complex remained unclear. We investigated the regulatory role of the serine cluster residing immediately adjacent to the HLH domain and of the serines in the NEMO/IKKγ-binding domain (NBD/γBD) in the C-terminal portion of IKKβ in MEFs deficient in IKKβ and IKKα and in yeast reconstitution system. We show that phosphorylation events at the C terminus of IKKβ can be divided into autophosphorylation of the serine cluster adjacent to the HLH domain and phosphorylation of the NBD/γBD. Autophosphorylation of the serine cluster occurs immediately after IKK activation and requires IKKγ. In MEFs, this autophosphorylation does not have the down-regulatory function on the IKK complex that was previously described (1). On the other hand, phosphorylation of the NBD/γBD regulates IKKγ-dependent phosphorylation of the T-loop activation domain in IKKβ and, hence, IKK complex activation. Our study suggests that, within the IKK complex, modulation of the NBD/γBD by IKKγ is upstream to the T-loop phosphorylation.

DNA-PKcs regulates a single-stranded DNA endonuclease activity of Artemis.

Human nuclease Artemis belongs to the metallo-beta-lactamase protein family. It acquires double-stranded DNA endonuclease activity in the presence of DNA-PKcs. This double-stranded DNA endonuclease activity is critical for opening DNA hairpins in V(D)J recombination and is thought to be important for processing overhangs during the nonhomologous DNA end joining (NHEJ) process. Here we show that purified human Artemis exhibits single-stranded DNA endonuclease activity. This activity is proportional to the amount of highly purified Artemis from a gel filtration column. The activity is stimulated by DNA-PKcs and modulated by purified antibodies raised against Artemis. Moreover, the divalent cation-dependence and sequence-dependence of this single-stranded endonuclease activity is the same as the double-stranded DNA endonuclease activity of Artemis:DNA-PKcs. These findings further expand the range of DNA substrates upon which Artemis and Artemis:DNA-PKcs can act. The findings are discussed in the context of NHEJ.