A novel pathogenic mitochondrial DNA variant m.4344T>C in tRNAGln causes developmental delay.

Mitochondrial diseases are a group of genetic diseases caused by mutations in mitochondrial DNA and nuclear DNA. However, the genetic spectrum of this disease is not yet complete. In this study, we identified a novel variant m.4344T>C in mitochondrial tRNAGln from a patient with developmental delay. The mutant loads of m.4344T>C were 95% and 89% in the patient's blood and oral epithelial cells, respectively. Multialignment analysis showed high evolutionary conservation of this nucleotide. TrRosettaRNA predicted that m.4344T>C variant would introduce an additional hydrogen bond and alter the conformation of the T-loop. The transmitochondrial cybrid-based study demonstrated that m.4344T>C variant impaired the steady-state level of mitochondrial tRNAGln and decreased the contents of mitochondrial OXPHOS complexes I, III, and IV, resulting in defective mitochondrial respiration, elevated mitochondrial ROS production, reduced mitochondrial membrane potential and decreased mitochondrial ATP levels. Altogether, this is the first report in patient carrying the m.4344T>C variant. Our data uncover the pathogenesis of the m.4344T>C variant and expand the genetic mutation spectrum of mitochondrial diseases, thus contributing to the clinical diagnosis of mitochondrial tRNAGln gene variants-associated mitochondrial diseases.

Stressful Life Events and Problem Gambling Among Chinese Lottery Gamblers: The Mediating Effects of Coping Strategies and Magical Thinking.

Problem gambling poses serious harm to individuals and societies worldwide. This study aims to investigate the relationship between stressful life events and problem gambling, and further explore the mediating role of coping strategies and magical thinking. Currently, the research on problem gambling is widely conducted worldwide. However, due to the unique characteristics of China's gambling industry, research on problem gambling conducted in the Chinese mainland has always been an underrepresented area in international gambling research. This study recruited participants from a province in central China, and data from 483 of them were ultimately analyzed. The data analysis results indicate that task-oriented coping, emotion-oriented coping, avoidance-oriented coping, and magical thinking all serve as mediators in the relationship between stressful life events and problem gambling. Emotion-oriented coping and magical thinking, avoidance-oriented coping and magical thinking, all serve as serial mediators in the relationship between stressful life events and problem gambling. Task-oriented coping and magical thinking did not act as serial mediators in this relationship. This study demonstrates that helping problem gamblers develop effective coping strategies and reduce their level of magical thinking is crucial for treating their problem gambling.

Peroxisome-based metabolic engineering for biomanufacturing and agriculture.

Subcellular compartmentalization of metabolic pathways plays a crucial role in metabolic engineering. The peroxisome has emerged as a highly valuable and promising compartment for organelle engineering, particularly in the fields of biological manufacturing and agriculture. In this review, we summarize the remarkable achievements in peroxisome engineering in yeast, the industrially popular biomanufacturing chassis host, to produce various biocompounds. We also review progress in plant peroxisome engineering, a field that has already exhibited high potential in both biomanufacturing and agriculture. Moreover, we outline various experimentally validated strategies to improve the efficiency of engineered pathways in peroxisomes, as well as prospects of peroxisome engineering.

A peroxisomal cinnamate:CoA ligase-dependent phytohormone metabolic cascade in submerged rice germination.

The mechanism underlying the ability of rice to germinate underwater is a largely enigmatic but key research question highly relevant to rice cultivation. Moreover, although rice is known to accumulate salicylic acid (SA), SA biosynthesis is poorly defined, and its role in underwater germination is unknown. It is also unclear whether peroxisomes, organelles essential to oilseed germination and rice SA accumulation, play a role in rice germination. Here, we show that submerged imbibition of rice seeds induces SA accumulation to promote germination in submergence. Two submergence-induced peroxisomal Oryza sativa cinnamate:CoA ligases (OsCNLs) are required for this SA accumulation. SA exerts this germination-promoting function by inducing indole-acetic acid (IAA) catabolism through the IAA-amino acid conjugating enzyme GH3. The metabolic cascade we identified may potentially be adopted in agriculture to improve the underwater germination of submergence-intolerant rice varieties. SA pretreatment is also a promising strategy to improve submerged rice germination in the field.

Downregulated FTO Promotes MicroRNA-155-mediated Inflammatory Response in Cerebral Ischemia/Reperfusion Injury.

This study aimed to elucidate the mechanism for alteration of m6A RNA modification in cerebral ischemia/reperfusion(I/R) injury and identify novel therapeutic targets. A rat cerebral I/R injury model was established by middle cerebral artery occlusion (MCAO) followed by reperfusion. Changes in m6A RNA modification were evaluated by colorimetric quantification. The expression of the m6A methyltransferases METTL3, METTL14, and WTAP, and the demethylases FTO and ALKBH5 were determined using qPCR and western blot analyses. FTO was overexpressed in brain tissues via intracerebroventricular injection of adenoviruses encoding FTO. The protective effect of FTO on m6A RNA modification and cerebral I/R injury was assessed. MeRIP assays were used to detect the impact of FTO overexpression on m6A modification of pri-miR-155; qPCR analysis was used to identify its maturation. Finally, the role of miR-155 overexpression in the protective effects of FTO on cerebral I/R injury was examined. m6A levels of total RNA were increased, and m6A methyltransferase FTO expression was decreased in post-I/R injury cerebral tissues. FTO overexpression reversed the increase in m6A RNA modification and attenuated cerebral I/R injury. Furthermore, FTO overexpression increased the m6A modification of pri-miR-155 and enhanced its maturation to form miR-155. Notably, miR-155 overexpression blunted FTO's protective effect against cerebral I/R injury. We propose that downregulation of FTO expression contributes to increased m6A RNA modification in cerebral I/R injury. FTO overexpression reverses increased total m6A RNA modification and exerts a protective effect against cerebral I/R injury via downregulating m6A modification of pri-miR-155 to inhibit its maturation process.

Bioinformatic analysis of short-chain dehydrogenase/reductase proteins in plant peroxisomes.

Peroxisomes are ubiquitous eukaryotic organelles housing not only many important oxidative metabolic reactions, but also some reductive reactions that are less known. Members of the short-chain dehydrogenase/reductase (SDR) superfamily, which are NAD(P)(H)-dependent oxidoreductases, play important roles in plant peroxisomes, including the conversion of indole-3-butyric acid (IBA) to indole-3-acetic acid (IAA), auxiliary ß-oxidation of fatty acids, and benzaldehyde production. To further explore the function of this family of proteins in the plant peroxisome, we performed an in silico search for peroxisomal SDR proteins from Arabidopsis based on the presence of peroxisome targeting signal peptides. A total of 11 proteins were discovered, among which four were experimentally confirmed to be peroxisomal in this study. Phylogenetic analyses showed the presence of peroxisomal SDR proteins in diverse plant species, indicating the functional conservation of this protein family in peroxisomal metabolism. Knowledge about the known peroxisomal SDRs from other species also allowed us to predict the function of plant SDR proteins within the same subgroup. Furthermore, in silico gene expression profiling revealed strong expression of most SDR genes in floral tissues and during seed germination, suggesting their involvement in reproduction and seed development. Finally, we explored the function of SDRj, a member of a novel subgroup of peroxisomal SDR proteins, by generating and analyzing CRISPR/Cas mutant lines. This work provides a foundation for future research on the biological activities of peroxisomal SDRs to fully understand the redox control of peroxisome functions.

(1E,4E)-1-(2-Nitro-phen-yl)-5-(2,6,6-trimethyl-cyclo-hex-1-en-1-yl)penta-1,4-dien-3-one.

In the title curcumin-ionone derivative, C(20)H(23)NO(3), the dihedral angle between the cyclo-hexene and benzene rings is 21.03 (8)°, with both double bonds in the inter-linking olefinic chain adopting E conformations. Two of the methyl-ene groups of the ß-ionone ring are disordered over two sets of sites with occupancy ratios of 0.50:0.50 and 0.60:0.40. In the crystal, mol-ecules are linked by weak C-H⋯O hydrogen bonds into zigzag chains extending along the b axis.

(1E,4E)-1-(Thio-phen-2-yl)-5-(2,6,6-trimethyl-cyclo-hex-1-en-1-yl)penta-1,4-dien-3-one.

In the title curcumin-ionone derivative, C(18)H(22)OS, the dihedral angle between the thia-zole ring and the mean plane through the cyclo-hexene ring is 5.16 (10)°. The mol-ecule has an E conformation for each of the olefinic bonds.

Protein phosphatase 2A (PP2A) holoenzymes regulate death-associated protein kinase (DAPK) in ceramide-induced anoikis.

The tumor suppressor, death-associated protein kinase (DAPK), is a Ca(2+)/calmodulin-regulated Ser/Thr kinase with an important role in regulating cytoskeletal dynamics. Autophosphorylation within the calmodulin-binding domain at Ser-308 inhibits DAPK catalytic activity. Dephosphorylation of Ser-308 by a previously unknown phosphatase enhances kinase activity and proteasome-mediated degradation of DAPK. In these studies, we identified two holoenzyme forms of protein phosphatase 2A (PP2A), ABalphaC and ABdeltaC, as DAPK-interacting proteins. These phosphatase holoenzymes dephosphorylate DAPK at Ser-308 in vitro and in vivo resulting in enhanced kinase activity of DAPK. The enzymatic activity of PP2A also negatively regulates DAPK levels by enhancing proteasome-mediated degradation of the kinase. Overexpression of wild type DAPK induces cell rounding and detachment in HEK293 cells; however, this effect is not observed following expression of an inactive DAPK S308E mutant. Finally, activation of DAPK by PP2A was found to be required for ceramide-induced anoikis. Together, our results provide a mechanism by which PP2A and DAPK activities control cell adhesion and anoikis.

Toll-like receptor 3 ligand dampens liver inflammation by stimulating Valpha 14 invariant natural killer T cells to negatively regulate gammadeltaT cells.

Valpha14 invariant natural killer T (Valpha14iNKT) cells are at the interface between the innate and adaptive immune responses and are thus critical for providing full engagement of host defense. We investigated the role of polyriboinosinic:polycytidylic acid (poly I:C), a replication-competent viral double-stranded RNA mimic and a specific agonist that recognizes the cellular sensor Toll-like receptor 3 (TLR3), in regulating Valpha14iNKT cell activation. We established for the first time that hepatic Valpha14iNKT cells up-regulate TLR3 extracellularly after poly I:C treatment. Notably, activation of TLR3-expressing hepatic Valpha14iNKT cells by a TLR3 ligand was suppressed by TLR3 deficiency. Our studies also revealed that Valpha14iNKT cell activation in response to poly I:C administration uniquely suppressed the accumulation and activation of intrahepatic gammadeltaT cells (but not natural killer cells) by inducing apoptosis. Furthermore, we established that activated hepatic Valpha14iNKT cells (via cytokines and possibly reactive oxygen species) influenced the frequency and absolute number of intrahepatic gammadeltaT cells, as evidenced by increased hepatic gammadeltaT cell accumulation in Valpha14iNKT cell-deficient mice after poly I:C treatment relative to wild-type mice. Thus, hepatic Valpha14iNKT cells and intrahepatic gammadeltaT cells are functionally linked on application of TLR3 agonist. Overall, our results demonstrate a novel and previously unrecognized anti-inflammatory role for activated hepatic Valpha14iNKT cells in negatively regulating intrahepatic gammadeltaT cell accumulation (probably through TLR3 signaling) and thereby preventing potentially harmful activation of intrahepatic gammadeltaT cells.

GammadeltaT cells initiate acute inflammation and injury in adenovirus-infected liver via cytokine-chemokine cross talk.

Emerging studies suggest an important role for the innate immune response in replication-defective adenovirus (Ad)-mediated acute liver toxicity. Specifically, classical innate immune cells (including NK cells, neutrophils, and Kupffer cells) have all been implicated in the development of Ad-mediated acute liver toxicity. The nonclassical innate immune T cell, the gammadeltaT cell, has been implicated in the pathophysiology of several viral infections that predominantly affect the mucosa and brain, but the specific role in the pathology of AdLacZ-mediated acute liver inflammation and injury as well as accompanying vector clearance is largely unknown. In the present study, we demonstrated that a CXCL9-CXCR3-dependent mechanism governed the accumulation of gammadeltaT cells in the livers of mice infected with Ad expressing the Escherichia coli LacZ gene (AdLacZ). We also showed a critical role for gammadeltaT cells in initiating acute liver toxicity after AdLacZ administration, driven in part by the ability of gammadeltaT cells to promote the recruitment of the conventional T cell, the CD8(+) T cell, into the liver. Furthermore, reduced hepatic injury in AdLacZ-infected gammadeltaT-cell-deficient mice was associated with lower hepatic levels of gamma interferon (IFN-gamma) and CXCL9, an IFN-gamma-inducible chemokine. Finally, our study highlighted a key role for IFN-gamma and CXCL9 cross talk acting in a feedback loop to drive the proinflammatory effects of gammadeltaT cells during AdLacZ-mediated acute liver toxicity. Specifically, intracellular IFN-gamma produced by activated hepatic gammadeltaT cells interacts with hepatocytes to mediate hepatic CXCL9 production, with the consequent accumulation of CXCR3-bearing gammadeltaT cells in the liver to cause acute liver damage without vector clearance.

Characterization of the role of TCR gammadelta in NK cell accumulation during viral liver inflammation.

Polyinosinic-polyctidylic acid (Poly I:C) is a viral RNA mimic that can induce immune responses similar to that seen during viral infection. Although poly I:C administration into mice is associated increased NK cell infiltrates in the liver, the mechanisms underlying increased hepatic NK cell accumulation in response to poly I:C administration are incompletely defined. In the current study, we have identified a novel and important role for gammadelta T cells in driving the accumulation and activation of NK cells in the liver during poly I:C-mediated viral liver infection. Specifically, NK cell accumulation but not activation in gammadelta T cell deficient mice following poly I:C administration was significantly attenuated in comparison to that seen in poly I:C-treated wildtype mice. The ability of gammadelta T cells to promote NK cell accumulation and activation in the liver may be virus-specific since NK cell accumulation in the liver was not altered by TCR gammadelta deficiency following adenovirus administration.

[Bilateral transpedicular balloon kyphoplasty for the treatment of osteoporotic vertebral compressive fractures].

OBJECTIVE: To compare clinical outcomes of bilateral transpedicular balloon kyphoplasty for the treatment of ordinary osteoporotic vertebral compressive fracture (OVCF) and severe osteoporotic vertebral compressive fracture. METHODS: From Junary 2009 to Febuary 2011, 60 patients (70 vertebrae) with osteoporotic vertebral compressive fracture were included. All patients were treated by bilateral transpedicular balloon kyphoplasty combined with postural reduction, including 10 males and 50 females aged from 59 to 90 years old with an average of 72.1 years old. In ordinary osteoporotic vertebral compressive fracture group, there were 38 patients (44 vertebrae) including 7 males and 31 females aged from 59 to 87 years old with an average of (71.8±6.1) years old. There were 6 patients with two vertebral fractures, 1 vertebra in T9, 5 vertebrae in T10, 7 vertebrae in T11, 13 vertebrae in T12, 9 vertebrae in L1, 4 vertebrae in L2, 4 vertebrae in L3, 1 vertebra in L4. While in severe osteoporotic vertebral compressive fracture group, there were 22 patients (26 vertebrae) including 3 males and 19 females aged from 63 to 90 years old with an average of (72.6±7.2) years old. There were 4 patients with two vertebral fractures, 1 vertebra in T9, 2 vertebrae in T10, 3 vertebrae in T11, 9 vertebrae in T12, 6 vertebrae in L1, 3 vertebrae in L2, 2 vertebrae in L3. Operative time, volume of bone cement injection, and vertebral height and changes of Cobb angle before and after operation were observed and compared. Postoperative average recovery rate of vertebral height and correct degree of Cobb angle were caculated and compared, VAS scoring were used to evaluate therapeutic effect. RESULTS: All operations were completed sucessfully, and pain were relieved at 72 h after operation. All patients were followed up from 6 to 13 months with an average of 10.1 months. Postoperative vertebral height, Cobb angle and VAS score were improved better than that of before operation (P<0.05). Operative time in ordinary group was shorter than severe group, while volume of bone cement injection was more than that of severe group. Average recovery rate of vertebral height and correct degree of Cobb angle in ordinarty group was better than that of in severe group (P<0.05). There was no significant differences between two groups in VAS scores before and after operation (P> 0.05). Three cases (3 vertebrae) ocurred bone cement leakage in ordinarty group, while 5 cases (5 vertebrae) ocurred bone cement leakage in severe group, and there was no meaning between two groups (P>0.05). CONCLUSION: Kyphoplasty could receive satisfied curative effect in treating ordinary and servere patients with osteoporotic vertebral compressive fracture, but recovery of vertebral height and correct degree of Cobb angle in ordinary gourp was better than that of in servere group.

[The clinical value of end plate rings in preventing subsidence of titanium cage in anterior cervical corpectomy and fusion surgery].

OBJECTIVE: To evaluate the clinical results of using end plate rings in preventing subsidence of titanium cage in anterior cervical corpectomy and fusion (ACCF) surgery. METHODS: The clinical data of 71 patients with cervical spondylotic myelopathy underwent ACCF in single segment from February 2008 to February 2011 were retrospectively analyzed. There were 38 males and 33 females, aged from 39 to 74 years old with a mean of 53.8 years. Thirty-three were used end plate rings and thirty-eight were not used (end plate rings group and no end plate ring group, respectively). The Japanese Orthopaedic Association (JOA) score, Odom's scale, imaging data were used to evaluate the clinical effects. Imaging data including Cobb angle of fusion segment, intervertebral height of anterior border (Da) and posterior border (Dp), the mean intervertebral height (Dm). RESULTS: All patients were followed up from 13 to 34 months with an average of 19.5 months. Between two groups, there was no significant difference in Cobb angle of fusion segment and the mean intervertebral height (Dm) before surgery and one week after surgery. Whereas, one year after surgery, the Cobb angle of end plate ring group was (9.4 ± 3.8) degrees, and contral group was (7.5 ± 3.9) degrees, which was significantly lower than that of end plate ring group. Meanwhile, the Dm of end plate ring group was (57.3 ± 2.2) mm, and no end ring group was (55.2 ± 2.6) mm which was significantly lower than that of end plate ring group. The subsidence in end plate ring group was 57.6%, and was 78.9% in no end plate ring group. There was no significant difference in JOA score before and after surgery between two groups. At 1 year after operation, 90.9% (30/33) got excellent or good results in end plate ring group, 89.5% (33/38) got excellent or good results in contral group. CONCLUSION: The use of end plate rings could not completely prevent the subsidence of titanium cage, however, which can decrease the occurrence rate of the subsidence and lessen its degree.

[Modified Stoppa approach in treatment of pelvic and acetabular fractures].

OBJECTIVE: To evaluate the modified Stoppa approach in treatment of pelvic and acetabular fractures. METHODS: From March 2010 to May 2012,16 patients with pelvic fractures and 7 patients with acetabutar fractures were treated by open reduction and internal fixation through the modified Stoppa approach,involving 18 males and 5 females with an average age of 39 years ranging from 17 to 65. By Tile classification, 16 cases of pelvic fractures included 1 case of B1, 2 of B2, 3 of B3, 4 of C1-1, 2 of C1-2, 2 of C1-3, and 1 of C2. By Letournel classification, 7 cases of acetabular fractures included 1 case of anterior column fractures, 1 of transverse fractures, 2 of type T, 1 of anterior column plus posterior transverse fractures, and 2 cases of both columns fractures. For 16 pelvic fractures, the modified Stoppa approach was used exclusively in 9 cases,in combination with the iliac fossa approach in 6 cases, and in combination with the posterior approach in 1 case. For 7 acetabular fractures, the modified Stoppa approach was used exclusively in 4 cases, in combination with the Kocher-Langenbeck approach in 2 cases, and in combination with the Kocher-Langenbeck and iliac fossa approaches in 1 case of both columns fractures. RESULTS: The average operation time was 130 min (50 to 350 min) and the blood loss averaged 320 ml (100 to 1200 ml). There were no operative complications. The reductions of the pelvic and acetabular fractures were all excellent and good. Twenty-one patients were followed-up from 4 to 24 months (averaged 8 months). The fractures were all healed,the fracture healing time was 2.5 to 5 months (means 3.2 months). Among them, 1 case occurrenced screw loosening, 1 case had mild limited of hip flexion, no case had plate breakage and lateral ventral syndrome. CONCLUSION: The modified Stoppa approach can be used to treat pelvic and acetabular fractures effectively, and it has advantages of easy manipulation and a low complication rate.

[Clinical application study of modified Moore classification in lower cervical spine injuries].

OBJECTIVE: To analyze the clinical application of modified Moore classification in lower cervical spine injuries. METHODS: Modified Moore classification was applied in the morphologic description of 200 patients (including 165 males, 35 females,age ranging from 19 to 88 years,with an average age of 52 years) with lower cervical spine injuries from August 2006 to March 2010, cervical spine injury severity score (quantification of stability) in combination with yes/no neurological injury status to classify their clinical diagnosis and management. The treatment was selected according to the fracture type, stability, compression injury of spinal cord or nerve roots, stability of ligamentous injury and other reference factors. According to the ASIA score, 130 cases with injury of spinal cord or nerve root (i.e. 6 cases in Grade A,13 cases in Grade B,43 cases in Grade C, 68 cases in Grade D); and 70 cases with no injury of spinal cord or nerve root. The ASIA score was applied in the evaluation of curative effect in cases with injury of spinal cord or nerve root. Radiodiagnostics was used to observe sequential measurement of cervical vertebrae and height in cases without spinal cord or nerve root injuries. RESULTS: The cervical spine injury distribution is that 35 cases of anterior, left, right lateral and posterior column injury; 33 cases of anterior column injury; 90 cases of anterior and posterior column injury; 5 cases of anterior, left lateral and posterior column injury; 3 cases of anterior, right lateral and posterior column injuries; 3 cases of anterior, left and right lateral column injuries; 2 cases of anterior and right lateral column injuries; 5 cases of anterior and left lateral column injury; 12 cases of posterior column injury; 7 cases of left lateral column injury; 5 cases of right lateral column injury. Surgery operation was given in 98 patients out of 200 cases. Non-surgical treatment was given to 102 patients (including 39 patients who are qualified to receive operation, but patient's relative required non -surgical treatment). Three cases of complete injury of spinal cord showed no recovery of the spinal cord function after operation, no change on the ASIA score, but pain and numbness of limb relieved slightly. Three non-surgical treatment cases showed no change after the treatment. Cases of incomplete injury of spinal cord showed certain recovery on spinal cord function after operation, and the ASIA score was raised 1.2 grades averagely. The ASIA score of cases of incomplete injury of spinal cord after non-surgical treatment was raised 0.3 grades averagely. The alignment and height of cervical vertebras were normal on post-operative radiodiagnostics in patients without injury of spinal cord or nerve root. CONCLUSION: According to modified Moore classification, when the stability quantification score is higher than or equal to 4, it indicated that the cervical vertebras are instability in lower cervical spine injuries. Surgery operation is required in higher score and less stability cases. Cases associated with neurological injury must receive surgery operation. Cases with stability quantification score equals to 3 and neurological injury should also receive surgery operation in general. Surgery operation is not required in cases of stability quantification equal to 3 and without neurological injury,or cases of stability quantification score lower than 3. Applying modified Moore classification in the treatment of lower cervical spine injuries is beneficial for the clinical standardization, diagnosis and treatment and receives satisfactory therapeutic effects.

Control of death-associated protein kinase (DAPK) activity by phosphorylation and proteasomal degradation.

Activation of death-associated protein kinase (DAPK) occurs via dephosphorylation of Ser-308 and subsequent association of calcium/calmodulin. In this study, we confirmed the existence of the alternatively spliced human DAPK-beta, and we examined the levels of DAPK autophosphorylation and DAPK catalytic activity in response to tumor necrosis factor or ceramide. It was found that DAPK is rapidly dephosphorylated in response to tumor necrosis factor or ceramide and then subsequently degraded via proteasome activity. Dephosphorylation and activation of DAPK are shown to temporally precede its subsequent degradation. This results in an initial increase in kinase activity followed by a decrease in DAPK expression and activity. The decline in DAPK expression is paralleled with increased caspase activity and cell apoptosis. These results suggest that the apoptosis regulatory activities mediated by DAPK are controlled both by phosphorylation status and protein stability.

Antisense depletion of death-associated protein kinase promotes apoptosis.

Death-associated protein kinases (DAPK) are serine/threonine protein kinases that have an important role in regulating cell death. In this study two antisense approaches were employed to down-regulate expression of the endogenous DAPK-alpha and DAPK-beta proteins. Transient expression of an antisense DAPK cDNA or antisense morpholino oligonucleotides in HeLa, 3T3, or primary human vascular smooth muscle cells demonstrate that decreased DAPK expression promotes a spontaneous, caspase-mediated apoptosis as evidenced by increased activities of caspases-3 and -9. Clonal HeLa cell lines with attenuated levels of DAPK expression, obtained following selection in the presence of antisense DAPK cDNA, are more sensitive to tumor necrosis factor-induced caspase-mediated apoptosis, and their sensitivity is inversely related to DAPK expression. In contrast, HeLa cells with reduced DAPK expression are moderately resistant to cell death induced by interferon-gamma. This finding is consistent with previous studies showing that DAPK has a role in promoting caspase-independent cell death. Together, these studies demonstrate that the cellular activities of DAPK are critical for antagonizing caspase-dependent apoptosis to promote cell survival under normal cell growth conditions.

A death-associated protein kinase (DAPK)-interacting protein, DIP-1, is an E3 ubiquitin ligase that promotes tumor necrosis factor-induced apoptosis and regulates the cellular levels of DAPK.

Death-associated protein kinase (DAPK) is a multi-domain Ser/Thr protein kinase with an important role in apoptosis regulation. In these studies we have identified a DAPK-interacting protein called DIP-1, which is a novel multi-RING finger protein. The RING finger motifs of DIP-1 have E3 ligase activity that can auto-ubiquitinate DIP-1 in vitro. In vivo, DIP-1 is detected as a polyubiquitinated protein, suggesting that the intracellular levels of DIP-1 are regulated by the ubiquitin-proteasome system. Transient expression of DIP-1 in HeLa cells antagonizes the anti-apoptotic function of DAPK to promote a caspase-dependent apoptosis. These studies also demonstrate that DAPK is an in vitro and in vivo target for ubiquitination by DIP-1, thereby providing a mechanism by which DAPK activities can be regulated through proteasomal degradation.