DEK is a nuclear phosphoprotein implicated in oncogenesis and autoimmunity and a major component of metazoan chromatin. The intracellular cues that control the binding of DEK to DNA and its pleiotropic functions in DNA-and RNA-dependent processes have remained mainly elusive so far. Our recent finding that the phosphorylation status of DEK is altered during death receptor-mediated apoptosis suggested a potential involvement of DEK in stress signaling. In this study, we show that in cells committed to die, a portion of the cellular DEK pool is extensively posttranslationally modified by phosphorylation and poly(ADP-ribosyl)ation. Through interference with DEK expression, we further show that DEK promotes the repair of DNA lesions and protects cells from genotoxic agents that typically trigger poly(ADP-ribose) polymerase activation. The posttranslational modification of DEK during apoptosis is accompanied by the removal of the protein from chromatin and its release into the extracellular space. Released modified DEK is recognized by autoantibodies present in the synovial fluids of patients affected by juvenile rheumatoid arthritis/juvenile idiopathic arthritis. These findings point to a crucial role of poly(ADP-ribosyl)ation in shaping DEK's autoantigenic properties and in its function as a promoter of cell survival.Human DEK is an abundant and highly conserved nuclear protein that has long been implicated in carcinogenesis and autoimmune disorders (for a review, see references 50 and 60). Originally isolated from a specific subtype of acute myeloid leukemia (55), the gene encoding DEK is expressed under the control of transcription factors E2F and YY1 (10, 49). High levels of DEK support cell immortalization and inhibit both senescence and apoptosis, as shown in cells infected with highrisk human papillomavirus E7 (1,62,63). DEK is also upregulated in a variety of aggressive human tumors, including retinoblastoma, colon and bladder cancer, and melanoma (e.g., see references 10, 19, 28, 30, and 37).In the nucleus, DEK is involved in a variety of DNA-and RNA-dependent processes, such as DNA replication (2), splice site recognition (51), and gene transcription. Here it can function as either an activator (9) or a repressor (16,20,45). The diversity of these effects is in line with DEK's described function as a possible regulator of chromatin architecture, which may affect genome activity at various levels in a highly contextdependent manner. In fact, DEK has been shown in vitro to be a modifier of DNA higher-order structure, acting in concert with topoisomerase I to introduce constrained positive supercoils in closed circular DNA plasmids and simian virus 40 (SV40) minichromosomes (24,25,58,59). Accordingly, DEK was shown to bind to DNA in a structure-specific rather than sequence-specific manner and to reduce the accessibility of chromatin to components of the replication machinery (2, 58). Beyond its effects on DNA topology, DEK can modulate the activity of other chromatin-associated proteins, such as P/CAF and p3...

Caspases were recently implicated in the functional impairment of the nuclear pore complex during apoptosis, affecting its dual activity as nucleocytoplasmic transport channel and permeability barrier. Concurrently, electron microscopic data indicated that nuclear pore morphology is not overtly altered in apoptotic cells, raising the question of how caspases may deactivate nuclear pore function while leaving its overall structure largely intact. To clarify this issue we have analyzed the fate of all known nuclear pore proteins during apoptotic cell death. Our results show that only two of more than 20 nuclear pore core structure components, namely Nup93 and Nup96, are caspase targets. Both proteins are cleaved near their N terminus, disrupting the domains required for interaction with other nucleoporins actively involved in transport and providing the permeability barrier but dispensable for maintaining the nuclear pore scaffold. Caspase-mediated proteolysis of only few nuclear pore complex components may exemplify a general strategy of apoptotic cells to efficiently disable huge macromolecular machines.Caspases are a family of highly specific cysteine proteases that play a central role in programmed cell death by apoptosis (1). By cleavage of a limited number of structural and regulatory proteins, they produce changes in cellular morphology and metabolism that are hallmarks of the apoptotic process. In the nucleus such changes include pronounced chromatin condensation, massive nucleosomal DNA fragmentation, and alterations in nuclear shape (2, 3). Recent reports also indicate that the regulated exchange of macromolecules between the nucleus and cytoplasm is impaired during apoptosis (4 -8).Regulated nucleocytoplasmic transport across the nuclear envelope occurs via nuclear pore complexes (NPCs), 2 channels of elaborate architecture that consist of multiple copies of about 30 different nucleoporins (9). Many nucleoporins are components of distinct subcomplexes that are arranged around the central pore channel in 8-fold rotational symmetry. The vertebrate Nup93 subcomplex, embedded within the NPC core, harbors Nup205, Nup188,. This subcomplex is believed to be an anchor site for the Nup62 subcomplex that resides close to the pore channel mid-axis and consists of Nup62, Nup58, and Nup54 (13,14). The Nup93 subcomplex furthermore is flanked on both sides by the Nup160 subcomplex, consisting of nine proteins, i.e. Nup160, Nup133, Nup107, Nup96, Nup75/85, Nup43, Nup37, Sec13, and Seh1 (12,[15][16][17][18]. Another nucleoporin, Nup98 (19,20), is not stably integrated in the Nup160 subcomplex but interacts with one of its components, Nup96 (16,21). Whether other NPC core components, namely Nup155 (22), NLP1/CG1 (23), Nup35, and Aladin (9) directly interact with any known subcomplex or might be part of yet another still needs to be investigated.These subcomplexes are thought to be anchored to the pore wall by direct or indirect interaction with transmembrane proteins. Two such pore wall transmembrane proteins, gp210 (2...

During apoptosis nuclear morphology changes dramatically due to alterations of chromatin architecture and cleavage of structural nuclear proteins. To characterize early events in apoptotic nuclear dismantling we have performed a proteomic study of apoptotic nuclei. To this end we have combined a cell-free apoptosis system with a proteomic platform based on the differential isotopic labeling of primary amines with N-nicotinoyloxy-succinimide. We exploited the ability of this system to produce nuclei arrested at different stages of apoptosis to analyze proteome alterations which occur prior to or at a low level of caspase activation. We show that the majority of proteins affected at the onset of apoptosis are involved in chromatin architecture and RNA metabolism. Among them is DEK, an architectural chromatin protein which is linked to autoimmune disorders. The proteomic analysis points to the occurrence of multiple PTMs in early apoptotic nuclei. This is confirmed by showing that the level of phosphorylation of DEK is decreased following apoptosis induction. These results suggest the unexpected existence of an early crosstalk between cytoplasm and nucleus during apoptosis. They further establish a previously unrecognized link between DEK and cell death, which will prove useful in the elucidation of the physiological function of this protein.

Alicyclic compounds with hydroxyl groups represent common structures in numerous natural compounds, such as terpenes and steroids. Their degradation by microorganisms in the absence of dioxygen may involve a COC bond ring cleavage to form an aliphatic intermediate that can be further oxidized. The cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) from denitrifying Azoarcus sp. strain 22Lin, grown on cyclohexane-1,2-diol as a sole electron donor and carbon source, is the first thiamine diphosphate (ThDP)-dependent enzyme characterized to date that cleaves a cyclic aliphatic compound. The degradation of cyclohexane-1,2-dione (CDO) to 6-oxohexanoate comprises the cleavage of a COC bond adjacent to a carbonyl group, a typical feature of reactions catalyzed by ThDP-dependent enzymes. In the subsequent NAD ؉ -dependent reaction, 6-oxohexanoate is oxidized to adipate. CDH has been purified to homogeneity by the criteria of gel electrophoresis (a single band at ϳ59 kDa; calculated molecular mass, 64.5 kDa); in solution, the enzyme is a homodimer (ϳ105 kDa; gel filtration). As isolated, CDH contains 0.8 ؎ 0.05 ThDP, 1.0 ؎ 0.02 Mg 2؉ , and 1.0 ؎ 0.015 flavin adenine dinucleotide (FAD) per monomer as a second organic cofactor, the role of which remains unclear. Strong reductants, Ti(III)-citrate, Na ؉ -dithionite, and the photochemical 5-deazaflavin/oxalate system, led to a partial reduction of the FAD chromophore. The cleavage product of CDO, 6-oxohexanoate, was also a substrate; the corresponding cyclic 1,3-and 1,4-diones did not react with CDH, nor did the cis-and trans-cyclohexane diols. The enzymes acetohydroxyacid synthase (AHAS) from Saccharomyces cerevisiae, pyruvate oxidase (POX) from Lactobacillus plantarum, benzoylformate decarboxylase from Pseudomonas putida, and pyruvate decarboxylase from Zymomonas mobilis were identified as the closest relatives of CDH by comparative amino acid sequence analysis, and a ThDP binding motif and a 2-fold Rossmann fold for FAD binding could be localized at the C-terminal end and central region of CDH, respectively. A first mechanism for the ring cleavage of CDO is presented, and it is suggested that the FAD cofactor in CDH is an evolutionary relict.Alicyclic compounds, such as steroids and terpenes, are widespread in nature. They are produced by plant cells as secondary metabolites and occur in fossil fuels. Microorganisms can convert these compounds to cellular metabolites under oxic and anoxic conditions. Their biodegradation proceeds via COC bond ring cleavage to form an aliphatic intermediate, which can be further degraded by ␤-oxidation. In aerobic bacteria, the cleavage of the cyclic compound is catalyzed by a NADPH-dependent, flavin-containing monooxygenase. For example, cyclohexanone is converted to ε-caprolactone in a Bayer-Villiger-type reaction (14). Subsequently, the lactone is hydrolyzed to 6-hydroxyhexanoate (63), followed by two NAD ϩ /NADP ϩ -dependent oxidation steps with adipate as the final product. In anaerobes, such as Pseudomonas sp. strain K601, cyclohexa...

Alicyclic alcohols are naturally occurring compounds wh ich can be degraded by microorgan isms via cleavage of the ring CC bond. DenitrifyingAzoarcus sp. strain 22Lin grows on cyclohexane-l .2-diol which serves as electron donor and carbon source. The diol is converted to cyclohexane-l.2-dione followed by hydrolysis to the corresponding semialdehyde and oxidation to adipate. The latter two reactions are catalyzed by the thiamine diphosphate-dependent llavoenzyme cyciohexane-1.2-dione hydrolase. the first a-ketolase known so far. Biochemical and structural properties of this new member of the thiamine diphosphate enzyme family will be presented.