Overexpressed TTC3 Protein Tends to be Cleaved into Fragments and Form Aggregates in the Nucleus.

Human tetratricopeptide repeat domain 3 (TTC3) is a gene on 21q22.2 within the Down syndrome critical region (DSCR). Earlier studies suggest that TTC3 may be an important regulator in individual development, especially in neural development. As an E3 ligase, TTC3 binds to phosphorylated Akt and silence its activity via proteasomal cascade. Several groups also reported the involvement of TTC3 in familial Alzheimer's disease recently. In addition, our previous work shows that TTC3 also regulates the degradation of DNA polymerase gamma and over-expressed TTC3 protein tends to form insoluble aggregates in cells. In this study, we focus on the solubility and intracellular localization of TTC3 protein. Over-expressed TTC3 tends to form insoluble aggregates over time. The proteasome inhibitor MG132 treatment resulted in more TTC3 aggregates in a short period of time. We fused the fluorescent protein to either terminus of the TTC3 protein and found that the intracellular localization of fluorescent signals are different between the N-terminal tagged and C-terminal tagged proteins. Western blotting revealed that the TTC3 protein is cleaved into fragments of different sizes at multiple sites. The N-terminal sub-fragments of TTC3 are prone to from nuclear aggregates and the TTC3 nuclear import is mediated by signals within the N-terminal 1 to 650 residues. Moreover, over-expressed TTC3 induced a considerable degree of cytotoxicity, and its N-terminal sub-fragments are more potent inhibitors of cell proliferation than full-length protein. Considering the prevalent proteostasis dysregulation in neurodegenerative diseases, these findings may relate to the pathology of such diseases.

Tetratricopeptide repeat domain 3 overexpression tends to form aggregates and inhibit ubiquitination and degradation of DNA polymerase γ.

Tetratricopeptide repeat (TPR) domain 3 (TTC3) is a protein that contains canonical RING finger and TPR motifs. It is encoded by the TTC3 gene located in the Down syndrome critical region (DSCR). In this study, we used a yeast two-hybrid assay to identify several proteins that physically interact with TTC3, including heat shock proteins and DNA polymerase γ (POLG). When TTC3 was overexpressed in mammalian cells, the ubiquitination of POLG was inhibited and its degradation slowed. High TTC3 protein expression led to the development of intracellular TTC3 aggregates, which also contained POLG. Co-expression with Hsp70 or placing the TTC3 gene under control of an inducible promoter alleviated the aggregation and facilitated POLG degradation. As a result of POLG's effects on aging processes, we propose that a copy number variant of the TTC3 may contribute to Down syndrome pathogenesis.

[Study of changes in extravascular lung water index of patients suffered from H7N9 avian influenza with acute respiratory distress syndrome].

OBJECTIVE: To analyze the characteristic of changes in extravascular lung water index (EVLWI) of H7N9 avian influenza patients who complicated with acute respiratory distress syndrome (ARDS), and to approach the relevance between EVLWI and severity, pulmonary oxygenation in patients with lung injury. METHODS: Four H7N9 avian influenza patients administered from April to June in 2013 in First Affiliated Hospital of Nanchang University were studied. The patients who suffered from severe ARDS were administered with low tide volume ventilation plus positive end-expiratory pressure (PEEP), namely protected ventilation strategy, with monitoring hemodynamic parameters and EVLWI through pulse-indicated continuous cardiac output (PiCCO) catheter. During ventilation, patients' parameters, such as PEEP, fraction of inspired oxygen (FiO2), arterial partial pressure of oxygen (PaO2), oxygenation index (PaO2/FiO2), cardiac index (CI), systemic vascular resistance index (SVRI), pulmonary vascular resistance index (PVRI), EVLWI, and central venous pressure (CVP) were collected. RESULTS: All 4 H7N9 avian influenza patients were complicated with ARDS, 2 patients were classified to severe ARDS and administered with comprehensive therapies, specially protected ventilation strategy; ventilation duration was 9 days and 30 days respectively, and PiCCO monitoring was 9 days and 21 days respectively. EVLWI of 2 patients on the 1st, 2nd, 3rd day was 10.0±3.2 ml/kg, 12.0±2.9 ml/kg, 14.0±4.2 ml/kg, and 24.0±6.7 ml/kg, 24.0±6.1 ml/kg, 23.0±5.8 ml/kg, respectively. As their conditions became better, patients' EVLWI decreased to 5.5±2.7 ml/kg and 7.0±3.0 ml/kg, respectively at weaning. PEEP and FiO2 of 2 patients were down-regulated, PaO2/FiO2 increased to 334±64 mm Hg and 142±53 mm Hg at weaning. However, no significant changes in CI, SVRI, PVRI and CVP in the 2 patients were observed. CONCLUSIONS: EVLWI increases when H7N9 avian influenza patients are complicated with severe ARDS. As the conditions get better, EVLWI returns to normal value gradually. There is relevance between the motive changes in EVLWI and severity of ARDS and pulmonary oxygenation.

[Effect of ulinastatin on the expression of heat shock protein 70 and NF-kappaB in lung tissue in rats with paraquat poisoned].

OBJECTIVE: To observe the expression levels of heat shock protein 70 (hsp70) and NF-kappaB p65 mRNA in lung tissue of acute paraquat (PQ) poisoning rats, and intervention effects of ulinastatin (UTI). METHODS: Seventy-two Sprague-Dawley (SD) rats were randomly divided into three groups: PQ poisoning group, UTI group and control group. The rats were exposed intragastrically to PQ at the dose of 80 mg/kg to establish a model of the rat acute lung injury. The UTI group was intervened by peritoneal injection with 10000 U/kg UTI in 30 minutes. On the 12, 24, 48, 72 h after exposure, myeloperoxidase (MPO) activity in lung tissue were detected. The expression of the NF-kappaB p65 mRNA and hsp70 mRNA in lung tissue was detected by the reverse transcription-PCR (RT-PCR). The lung pathological changes of rats were observed. RESULTS: The degree of lung injury in PQ group and UTI group was higher than that in control group. But in UTI group the degree of lung injury was lower than PQ group. MPO activity in the lung tissues in PQ group was (31.72 +/- 6.42), (56.23 +/- 8.63), (87.21 +/- 10.02) and (107.21 +/- 13.52) micro/g in 12, 24, 48 and 72 h, respectively which was significantly higher than that [(11.38 +/- 1.25) micro/g] in control group (P < 0.01). MPO activity in the lung tissues in UTI group was (15.65 +/- 3.21), (35.98 +/- 5.74), (59.33 +/- 9.65) and (71.25 +/- 10.58) micro/g in 12, 24, 48 and 72 h, respectively which was significantly lower than those in PQ group (P < 0.01). The expression levels of NF-kappaB p65 mRNA of lung tissues in UTI group in 12, 24, 48 and 72 h were 0.3288 +/- 0.0147, 0.5337 +/- 0.0328, 0.7357 +/- 0.0424 and 0.7547 +/- 0.0905, respectively, which were significantly lower that those (0.4185 +/- 0.0294, 0.8532 +/- 0.0841, 0.9554 +/- 0.0975 and 1.0094 +/- 0.0703) in PQ group (P < 0.01). hsp70 mRNA expression levels in 12, 24, 48 and 72 h of the UTI group were 0.5193 +/- 0.0254, 0.8289 +/- 0.0606, 0.7566 +/- 0.0277 and 0.4873 +/- 0.0105, respectively, which were significantly higher than those (0.3897 +/- 0.0125, 0.5904 +/- 0.0186, 0.4007 +/- 0.0237 and 0.2293 +/- 0.0137) in PQ group (P < 0.01). CONCLUSION: The expression levels of hsp70 mRNA and NF-kappaB p65 mRNA of rats after intoxication increased significantly. UTI can protect the lung tissues by elevating the expression of hsp70 and reducing the expression of NF-kappaB in the lung tissues of rats with acute paraquat poisoning.