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1.  Autophagy-related gene 7 is downstream of heat shock protein 27 in the regulation of eye morphology, polyglutamine toxicity, and lifespan in Drosophila 
Background
Autophagy and molecular chaperones both regulate protein homeostasis and maintain important physiological functions. Atg7 (autophagy-related gene 7) and Hsp27 (heat shock protein 27) are involved in the regulation of neurodegeneration and aging. However, the genetic connection between Atg7 and Hsp27 is not known.
Methods
The appearances of the fly eyes from the different genetic interactions with or without polyglutamine toxicity were examined by light microscopy and scanning electronic microscopy. Immunofluorescence was used to check the effect of Atg7 and Hsp27 knockdown on the formation of autophagosomes. The lifespan of altered expression of Hsp27 or Atg7 and that of the combination of the two different gene expression were measured.
Results
We used the Drosophila eye as a model system to examine the epistatic relationship between Hsp27 and Atg7. We found that both genes are involved in normal eye development, and that overexpression of Atg7 could eliminate the need for Hsp27 but Hsp27 could not rescue Atg7 deficient phenotypes. Using a polyglutamine toxicity assay (41Q) to model neurodegeneration, we showed that both Atg7 and Hsp27 can suppress weak, toxic effect by 41Q, and that overexpression of Atg7 improves the worsened mosaic eyes by the knockdown of Hsp27 under 41Q. We also showed that overexpression of Atg7 extends lifespan and the knockdown of Atg7 or Hsp27 by RNAi reduces lifespan. RNAi-knockdown of Atg7 expression can block the extended lifespan phenotype by Hsp27 overexpression, and overexpression of Atg7 can extend lifespan even under Hsp27 knockdown by RNAi.
Conclusions
We propose that Atg7 acts downstream of Hsp27 in the regulation of eye morphology, polyglutamine toxicity, and lifespan in Drosophila.
doi:10.1186/1423-0127-19-52
PMCID: PMC3483682  PMID: 22621211
Atg7; Hsp27; Neurodegeneration; Lifespan; Drosophila
2.  Suppressive Regulation of KSHV RTA with O-GlcNAcylation 
Background
The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is a molecular switch that initiates a productive replication of latent KSHV genomes. KSHV RTA (K-RTA) is composed of 691 amino acids with high Ser and Thr content (17.7%), but to what extent these Ser and Thr are modified in vivo has not been explored.
Methods
By using tandem mass spectrometric analysis of affinity-purified FLAG tagged K-RTA, we sought to identify Ser and Thr residues that are post-translationally modified in K-RTA.
Results
We found that K-RTA is an O-GlcNAcylated protein and Thr-366/Thr-367 is the primary motif with O-GlcNAcylation in vivo. The biological significance of O-GlcNAc modified Thr-366 and Thr-367 was assessed by site-specific amino acid substitution. Replacement of Thr with Ala at amino acid 366 or 367 caused a modest enhancement of K-RTA transactivation activity in a luciferase reporter assay and a cell model for KSHV reactivation. By using co-immunoprecipitation coupled with western blot analysis, we showed that the capacity of K-RTA in associating with endogenous PARP1 was significantly reduced in the Thr-366/Thr-367 O-GlcNAc mutants. PARP1 is a documented negative regulator of K-RTA that can be ascribed by the attachment of large negatively charged polymer onto K-RTA via PARP1's poly (ADP-ribose) polymerase activity. In agreement, shRNA-mediated depletion of O-GlcNAc transferase (OGT) in KSHV infected cells augmented viral reactivation and virus production that was accompanied by diminished K-RTA and PARP1 complexes.
Conclusions
KSHV latent-lytic switch K-RTA is modified by cellular O-GlcNAcylation, which imposes a negative effect on K-RTA transactivation activity. This inhibitory effect involves OGT and PARP1, two nutritional sensors recently emerging as chromatin modifiers. Thus, we speculate that the activity of K-RTA on its target genes is continuously checked and modulated by OGT and PARP1 in response to cellular metabolic state.
doi:10.1186/1423-0127-19-12
PMCID: PMC3395832  PMID: 22300411
KSHV; K-RTA; O-GlcNAcylation; PARP1; Polycomb group (PcG) complex

Results 1-2 (2)