DNA damage response signaling does not trigger redistribution of SAMHD1 to nuclear foci.

SAMHD1 (Sterile alpha motif and histidine-aspartic acid (HD) domain containing protein 1) is a deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase) that restricts viral replication in infected cells. This protein is also involved in DNA repair by assisting in DNA end resection by homologous recombination (HR) after DNA double-strand break (DSB) induction with camptothecin (CPT) or etoposide (ETO). We showed that a monoclonal anti-SAMHD1 antibody produced against the full-length protein detected an unspecific 50 kDa protein that colocalized with dot-like structures after CPT treatment in HeLa cells. In contrast, a polyclonal anti-SAMHD1 antibody raised against the N-terminus of this protein specifically detected SAMHD1, as shown in Jurkat, HAP1KO and HEK293T SAMHD1-siRNA cell lysates compared with their respective controls. Our findings showed that SAMHD1 is not localized in dot-like structures under DSB induction in HeLa cells.

Uncovering Notch pathway in the parasitic flatworm Schistosoma mansoni.

Several signaling molecules that govern development in higher animals have been identified in the parasite Schistosoma mansoni, including the transforming growth factor ß, protein tyrosine kinases, nuclear hormone receptors, among others. The Notch pathway is a highly conserved signaling mechanism which is involved in a wide variety of developmental processes including embryogenesis and oogenesis in worms and flies. Here we aimed to provide the molecular reconstitution of the Notch pathway in S. mansoni using the available transcriptome and genome databases. Our results also revealed the presence of the transcripts coded for SmNotch, SmSu(H), SmHes, and the gamma-secretase complex (SmNicastrin, SmAph-1, and SmPen-2), throughout all the life stages analyzed. Besides, it was observed that the viability and separation of adult worm pairs were not affected by treatment with N-[N(3,5)-difluorophenacetyl)-L-Alanyl]-S-phenylglycine t-butyl ester (DAPT), a Notch pathway inhibitor. Moreover, DAPT treatment decreased the production of phenotypically normal eggs and arrested their development in culture. Our results also showed a significant decrease in SmHes transcript levels in both adult worms and eggs treated with DAPT. These results provide, for the first time, functional validation of the Notch pathway in S. mansoni and suggest its involvement in parasite oogenesis and embryogenesis. Given the complexity of the Notch pathway, further experiments shall highlight the full repertoire of Notch-mediated cellular processes throughout the S. mansoni life cycle.

The F-box protein FBXO25 promotes the proteasome-dependent degradation of ELK-1 protein.

FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens.

Investigation on the 19S ATPase proteasome subunits (Rpt1-6) conservation and their differential gene expression in Schistosoma mansoni.

The ubiquitin-proteasome system is responsible for degradation of the majority of intracellular proteins in eukaryotic cells. The 26S proteasome proteolytic complex is composed of a 20S core particle responsible for protein degradation and the 19S lid which plays a role in the recognition of polyubiquitinated substrates. The 19S regulatory particle (Rps) is composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. In this study, we analyzed the expression profile of 19S Rpt subunits in the larvae and adult stage of the Schistosoma mansoni life cycle. Conventional reverse transcriptase polymerase chain reaction (RT-PCR) revealed that the majority of the 19S Rpt subunits amplified at the expected molecular masses for various investigated stages. In addition, SmRpt1, SmRpt2, and SmRpt6 transcript levels were increased in 3 h-cultured schistosomula and reasonably maintained until 5 h in culture, as revealed by qRT-PCR. Phylogenetic analysis of 19S Rpt subunits showed high structural conservation in comparison to other Rpt orthologues. The mRNA expression profile of 19S Rpt subunits did not correlate with 26S proteasome proteolytic activity as judged by a (14)C-casein-degrading assay, in the early cultured schistosomula. Taken together, these results revealed a differential expression profile for 19S Rpt subunits whose transcript levels could not be directly associated to 26S proteasome activity.