Biomedical subjects

Noreen Williams

Publications and source records attributed to Noreen Williams.

6 recordsLinked to original sources

PubMed2006

ATP synthase is responsible for maintaining mitochondrial membrane potential in bloodstream form Trypanosoma brucei.

The mitochondrion of Trypanosoma brucei bloodstream form maintains a membrane potential, although it lacks cytochromes and several Krebs cycle enzymes. At this stage, the ATP synthase is present at reduced, although significant, levels. To test whether the ATP synthase at this stage is important for maintaining the mitochondrial membrane potential, we used RNA interference (RNAi) to knock down the levels of the ATP synthase by targeting the F1-ATPase alpha and beta subunits. RNAi-induced cells grew significantly slower than uninduced cells but were not morphologically altered. RNAi of the beta subunit decreased the mRNA and protein levels for the beta subunit, as well as the mRNA and protein levels of the alpha subunit. Similarly, RNAi of alpha subunit decreased the alpha subunit transcript and protein levels, as well as the beta-subunit transcript and protein levels. In contrast, alpha and beta RNAi knockdown resulted in a 60% increase in the F0 complex subunit 9 protein levels without a significant change in the steady-state transcript levels of this subunit. The F0-32-kDa subunit protein expression, however, remained stable throughout induction of RNAi for alpha or beta subunits. Oligomycin-sensitive ATP hydrolytic and synthetic activities were decreased by 43 and 44%, respectively. Significantly, the mitochondrial membrane potential of alpha and beta RNAi cells was decreased compared to wild-type cells, as detected by MitoTracker Red CMXRos fluorescence microscopy and flow cytometry. These results support the role of the ATP synthase in the maintenance of the mitochondrial membrane potential in bloodstream form T. brucei.

Animals↗

PubMed2004

TcRRMs and Tcp28 genes are intercalated and differentially expressed in Trypanosoma cruzi life cycle.

The identification and characterization of RNA binding proteins in Trypanosoma cruzi are particularly relevant as they play key roles in the regulatory mechanisms of gene expression. In this work, we have identified coding sequences for the proteins, named TcRRM1 and TcRRM2, in the EST database generated by the T. cruzi genomic initiative. TcRRM1 and TcRRM2 contain two RNA binding domains (RRM) and are very similar to two Trypanosoma brucei RNA binding proteins previously reported, Tbp34 and Tbp37, and to a not yet annotated ORF in Leishmania major genome project. The T. cruzi RRM genes are organized in tandem, alternating with copies of Tcp28, a gene of unknown function. However, TcRRM transcript accumulation is higher in the spheromastigote stage, while Tcp28 transcripts accumulate more in the trypomastigote stage suggesting developmental regulation.

Amino Acid Sequence↗

PubMed2003

A novel type of single-stranded nucleic acid binding protein recognizing a highly frequent motif in the intergenic regions of Trypanosoma cruzi.

Regulation of gene expression in trypanosomatids is not yet well understood. Genes are organized in long polycistronic transcriptional units separated by intergenic regions that may contain the signaling information for nucleic acid processing. Poly-dinucleotides are frequent in these regions and have been proposed to be involved in regulation of gene expression. Previously, we have reported that [dT-dG] are highly frequent, asymmetrically strand distributed, and constitute targets for specific protein binding [Biochem. Biophys. Res. Commun. 287 (2001) 98]. Here, we present the purification and characterization of a new type of single stranded nucleic acid binding protein (Tc38) that recognizes specifically the motif poly[dT-dG] in this parasite. The protein has a deduced molecular weight of 38kDa and its salient characteristics include an isoelectric point of 9.34, a high frequency of Ser, Leu, and di-amino acids. Neither compositional nor architectural conserved domains could be detected in database searches. Recombinant Tc38 was expressed as a GST fusion protein, purified, and used to analyze target specificity by electrophoretic mobility shift assays. The unusual characteristics of the protein together with the peculiar features of the specific nucleic acid target suggest the existence of a novel event that may be involved in the mechanisms of gene expression in trypanosomatids.

Amino Acid Motifs↗

PubMed2003

Stage-specific translational efficiency and protein stability regulate the developmental expression of p37, an RNA binding protein from Trypanosoma brucei.

We have previously characterized two novel RNA binding proteins, p34 and p37, from Trypanosoma brucei. Their sequences do not show significant homology to other proteins but are highly homologous to one another. The p34 and p37 proteins are developmentally regulated, with p34 the predominant protein in the procyclic stage and p37 nearly exclusively expressed in the bloodstream cells. In vivo metabolic labeling of procyclic cells showed that p34 and p37 were differentially translated, with levels of p34 approximately fourfold higher than p37. The newly synthesized p34 and p37 exhibited differential stability in the procyclic stage. In vitro analysis confirmed this observation and further suggested that this differential stability may be due to a trypsin-like cysteine protease activity in procyclic extracts that selectively degraded the p37 protein. Taken together, these results indicate that the developmental regulation of the T. brucei RNA binding protein, p37, occurs at both translational and post-translational levels.

Animals↗

PubMed2002

Two novel RNA binding proteins from Trypanosoma brucei are associated with 5S rRNA.

We have previously reported the identification of two closely related RNA binding proteins from Trypanosoma brucei which we have termed p34 and p37. The predicted primary structures of the two proteins are highly homologous with one major difference, an 18-amino-acid insert in the N-terminal region of p37. These two proteins have been localized to the nucleus based on immunofluorescence microscopy. To gain insight into their function, we have utilized UV crosslinking, coimmunoprecipitation, and sucrose density gradients to identify T. brucei RNA species that associate with p34 and p37. These experiments have demonstrated a specific interaction of both p34 and p37 with the 5S ribosomal RNA and indicate that other RNA species are unlikely to be specifically bound. This suggests a role for p34 and p37 in the import and/or assembly pathway of T. brucei 5S rRNA in ribosome biogenesis.

Animals↗

PubMed2002

Two families of RNA binding proteins from Trypanosoma brucei associate in a direct protein-protein interaction.

We have previously reported the identification of two closely related RNA binding proteins from Trypanosoma brucei, termed p34 and p37. The predicted primary structures of the two proteins are highly homologous with one major difference, an 18 amino acid insertion in the N-terminal region of p37. These two proteins are localized to the nucleus based on immunofluorescence microscopy. Recently, we have shown that p34 and p37 interact with T. brucei 5S rRNA. In order to gain further insight into their function, we have utilized protein affinity chromatography and immune capture approaches to identify T. brucei proteins which associate with p34 and p37. We demonstrate here an interaction of both p34 and p37 with the NOPP44/46 proteins, identified in T. brucei as a family of tyrosine-phosphorylated RNA binding proteins primarily localized to the nucleolus. This interaction was mapped to the RNA-binding region of p34/p37 and an acidic region of NOPP44/46 by protein affinity chromatography using recombinant deletion constructs of p34 and p37 and yeast two-hybrid analysis. These data may suggest a role for p34 and p37 and NOPP44/46 in the import and/or assembly pathway of T. brucei 5S rRNA in ribosome biogenesis.

Animals↗