Mice deficient in Epg5 exhibit selective neuronal vulnerability to degeneration.

The molecular mechanism underlying the selective vulnerability of certain neuronal populations associated with neurodegenerative diseases remains poorly understood. Basal autophagy is important for maintaining axonal homeostasis and preventing neurodegeneration. In this paper, we demonstrate that mice deficient in the metazoan-specific autophagy gene Epg5/epg-5 exhibit selective damage of cortical layer 5 pyramidal neurons and spinal cord motor neurons. Pathologically, Epg5 knockout mice suffered muscle denervation, myofiber atrophy, late-onset progressive hindquarter paralysis, and dramatically reduced survival, recapitulating key features of amyotrophic lateral sclerosis (ALS). Epg5 deficiency impaired autophagic flux by blocking the maturation of autophagosomes into degradative autolysosomes, leading to accumulation of p62 aggregates and ubiquitin-positive inclusions in neurons and glial cells. Epg5 knockdown also impaired endocytic trafficking. Our study establishes Epg5-deficient mice as a model for investigating the pathogenesis of ALS and indicates that dysfunction of the autophagic-endolysosomal system causes selective damage of neurons associated with neurodegenerative diseases.

PDCD5 interacts with p53 and functions as a positive regulator in the p53 pathway.

The tumor suppressor p53 is at the hub of cellular signaling networks that are activated by stress signals including DNA damage. In the present study, we showed that programmed cell death 5 (PDCD5) bound to p53 by glutathione S-transferase (GST)-pulldown, co-immunoprecipitation and co-localization assays. PDCD5 enhanced the stability of p53 by antagonizing Mdm2-induced p53 ubiquitination, nuclear export and proteasomal degradation. We also found that PDCD5 could dissociate the interaction between p53 and Mdm2 and interact with Mdm2 directly to promote its degradation. In cells with or without induction of DNA damage, knockdown of PDCD5 by RNA interference decreased the p53 phosphorylation at Ser9, 20 and 392 residues, as well as the expression of p21 protein. Additionally, chromatin immunoprecipitation assays showed an up-regulated association of PDCD5 at the p53BS2 site of the p21 promoter during DNA damage. Cell cycle analysis also indicated that PDCD5 was required in G1 phase cell arrest during DNA damage. In summary, PDCD5 may contribute to maintain a basal pool of p53 proteins in unstressed conditions, but upon DNA damage it functions as a co-activator of p53 to regulate transcription and cell cycle arrest.

Impaired Coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN is a neurodegenerative disease with unresolved pathophysiology. Previously, we observed reduced Coenzyme A levels in a Drosophila model for PKAN. Coenzyme A is required for acetyl-Coenzyme A synthesis and acyl groups from the latter are transferred to lysine residues of proteins, in a reaction regulated by acetyltransferases. The tight balance between acetyltransferases and their antagonistic counterparts histone deacetylases is a well-known determining factor for the acetylation status of proteins. However, the influence of Coenzyme A levels on protein acetylation is unknown. Here we investigate whether decreased levels of the central metabolite Coenzyme A induce alterations in protein acetylation and whether this correlates with specific phenotypes of PKAN models. We show that in various organisms proper Coenzyme A metabolism is required for maintenance of histone- and tubulin acetylation, and decreased acetylation of these proteins is associated with an impaired DNA damage response, decreased locomotor function and decreased survival. Decreased protein acetylation and the concurrent phenotypes are partly rescued by pantethine and HDAC inhibitors, suggesting possible directions for future PKAN therapy development.

Prognostic significance of downregulated expression of programmed cell death 5 in chondrosarcoma.

BACKGROUND AND OBJECTIVES: Programmed Cell Death 5 (PDCD5) is a novel apoptosis-related gene and deregulation of PDCD5 is involved in tumorigenicity. This study was designed to investigate the expression level of PDCD5 and to clarify its clinical significance in chondrosarcoma. METHODS: The mRNA and protein levels of PDCD5 in chondrosarcoma and matched corresponding non-tumor tissues were evaluated by real-time PCR and Western blot, respectively. The expression of PDCD5 protein was investigated by immunohistochemistry assays in chondrosarcoma, and its association with clinicopathologic factors and overall survival was also analyzed. RESULTS: The mRNA and protein levels of PDCD5 were significantly decreased in chondrosarcoma compared with corresponding non-tumor tissues. Low expression of PDCD5 protein was 61.8% (21/34) in chondrosarcomas, as compared 12.5% (1/8) in normal bones, as well as compared 23.5% (4/17) in benign cartilage tumors. PDCD5 expression was correlated with anatomical location and histological grade. The survival rate of patients with low-PDCD5 tumors was lower than that of patients with high-PDCD5 tumors. Multivariate analysis revealed that the downregulated expression of PDCD5 was an independent prognostic factor for overall survival. CONCLUSIONS: PDCD5 is downregulated in chondrosarcoma and might be an independent prognostic factor for overall survival of chondrosarcoma patients.

[Generation and characterization of monoclonal antibody against human PIK3IP1].

AIM: To obtain monoclonal antibody against PIK3IP1 for further study of the structure and biological function of PIK3IP1 protein. METHODS: BALB/c mice were immunized with recombinant GST-PIK3IP1(62-168), Hybridoma cell lines secreting monoclonal antibodies against PIK3IP1 were screened by regular cell fusion and subcloning approach. The specificities of the monoclonal antibody was determined by ELISA, Western blot and Immunofluorescence assay. RESULTS: One hybridoma cell line (5C6) stable in secreting specific monoclonal antibody was successfully obtained. The subclass of IgG belonged to IgG1. The ascite titers of this monoclonal antibody reached 1:10(7). It could specifically bind to recombinant GST-PIK3IP1(62-168); protein and overexpressed PIK3IP1 and variant PIK3IP1-v1 proteins proved by Western blot. This antibody failed to react with E.coli lysates and glutathione S transferase (GST). At the same time, endogenous PIK3IP1 was not detected using 5C6 antibody. Immunofluorescence results revealed that overexpressed PIK3IP1 and variant PIK3IP1-v1 protein located in cytoplasm and distributed in fleck manner. CONCLUSION: Monoclonal antibody against PIK3IP1 with high titer and specificity has been successfully generated, which could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties of PIK3IP1.

Recombinant human PDCD5 sensitizes chondrosarcomas to cisplatin chemotherapy in vitro and in vivo.

Clinical management of chondrosarcoma remains a challenging problem, largely due to the toxicity and resistance of this tumor to conventional chemotherapy. Programmed Cell Death 5 (PDCD5) is a protein that accelerates apoptosis in different cell types in response to various stimuli, and has been shown to be down-regulated in many cancer tissues. In this study, mRNA and protein levels of PDCD5 were found to be up-regulated in cisplatin-treated SW1353 chondrosarcoma cells compared with untreated cells. Recombinant human PDCD5 (rhPDCD5) was also shown to sensitize chondrosarcoma cells to cisplatin-based chemotherapy, with inhibition of cell growth and apoptosis detected both in vitro and in vivo. Increased expression of Bax and decreased expression of Bcl-2 were also observed, along with release of cytochrome c from mitochondria into the cytosol. Additionally, cleavage of caspase-9 and caspase-3, as well as the cleavage of poly (ADP-ribose) polymerase (PARP), were detected, suggesting that sensitization of chondrosarcoma cells involves the intrinsic mitochondrial apoptosis pathway. In vivo, the treatment of a xenograft model of chondrosarcoma with rhPDCD5 and cisplatin significantly inhibited tumor cell proliferation and induced apoptosis compared to treatment with cisplatin alone. Overall, these data provide a theoretical basis for the administration of rhPDCD5 and cisplatin for the treatment of patients with chondrosarcoma.

Structure-function correlation of human programmed cell death 5 protein.

Human programmed cell death 5 (PDCD5) is a translocatory protein playing an important role in the apoptotic process of cells. Although there are accumulated data about PDCD5 function, the correlation of the structure with the function of PDCD5 has not been investigated. Here, we report the studies of structure-function relationship of PDCD5 by multidimensional NMR methods and by FACScan flow cytometer and fluorescence microscope. The 3D structure of intact PDCD5 and the internal motions of PDCD5 have been determined. PDCD5 has a compact core structure of low flexibility with two mobile alpha-helices at N-terminal region and a flexible unstructured C-terminal region. The flow cytometry and internalization measurements of different PDCD5 fragments indicate that the charged residues are crucial for the ability of apoptosis-promoting and cell translocation of the protein. Combined analyses reveal a fact that the regions that seem to be most involved in the function also are more flexible in PDCD5.

PDCD5 interacts with Tip60 and functions as a cooperator in acetyltransferase activity and DNA damage-induced apoptosis.

Tip60 is a histone acetyltransferase (HAT) involved in the acetyltransferase activity and the cellular response to DNA damage. Here, we show that programmed cell death 5 (PDCD5), a human apoptosis-related protein, binds to Tip60 and enhances the stability of Tip60 protein in unstressed conditions. The binding amount of PDCD5 and Tip60 is significantly increased after UV irradiation. Further, PDCD5 enhances HAT activity of Tip60 and Tip60-dependent histone acetylation in both basal and UV-induced levels. We also find that PDCD5 increases Tip60-dependent K120 acetylation of p53 and participates in the p53-dependent expression of apoptosis-related genes, such as Bax. Moreover, we demonstrate the biological significance of the PDCD5-Tip60 interaction; that is, they function in cooperation to accelerate DNA damage-induced apoptosis and knockdown of PDCD5 or Tip60 impairs their apoptosis-accelerating activity, mutually. Consistent with this, PDCD5 levels increase significantly on DNA damage in U2OS cells, as does Tip60. Together, our findings indicate that PDCD5 may play a dual role in the Tip60 pathway. Specifically, under normal growth conditions, PDCD5 contributes to maintaining a basal pool of Tip60 and its HAT activity. After DNA damage, PDCD5 functions as a Tip60 coactivator to promote apoptosis.

[Preparation and identification of monoclonal antibodies against human programmed cell death 10 (PDCD10)].

OBJECTIVE: To obtain monoclonal antibodies against programmed cell death 10 (PDCD10) for further study of the structure and function of PDCD10 protein. METHODS: Balb/c mice were immunized with recombinant PDCD10, hybridoma cell lines secreting monoclonal antibodies against PDCD10 were screened by regular cell fusion and subcloning approach. The specificities of these monoclonal antibodies were determined by ELISA, Western blotting and Immunofluorescence assay. RESULTS: Three hybridoma cell lines (5G1, 4F7 and 3H5) stable in secreting specific monoclonal antibodies were successfully obtained. Subclass of IgG belonged to IgG1 (4F7 and 5G1) and IgG2b (3H5), respectively. The ascite titers of these monoclonal antibodies reached 1:10(7). They could specifically bind to recombinant PDCD10 and endogenous and overexpressed PDCD10 proteins proved by ELISA and Western blotting. They failed to react with E.coli lysates and glutathione S-transferase (GST). In addition, these three monoclonal antibodies could recognize different epitopes of PDCD10 proteins assessed by immune fluorescence competitive binding assay. Both endogenous and overexpressed PDCD10 protein mainly located in the nucleus. CONCLUSION: Monoclonal antibodies against PDCD10 with high titers and specificity have been successfully prepared, which has laid the foundation for further study of PDCD10 protein.