Biomedical subjects

Emily Hodges

Publications and source records attributed to Emily Hodges.

5 recordsLinked to original sources

PubMed2006

NovelFam3000--uncharacterized human protein domains conserved across model organisms.

BACKGROUND: Despite significant efforts from the research community, an extensive portion of the proteins encoded by human genes lack an assigned cellular function. Most metazoan proteins are composed of structural and/or functional domains, of which many appear in multiple proteins. Once a domain is characterized in one protein, the presence of a similar sequence in an uncharacterized protein serves as a basis for inference of function. Thus knowledge of a domain's function, or the protein within which it arises, can facilitate the analysis of an entire set of proteins. DESCRIPTION: From the Pfam domain database, we extracted uncharacterized protein domains represented in proteins from humans, worms, and flies. A data centre was created to facilitate the analysis of the uncharacterized domain-containing proteins. The centre both provides researchers with links to dispersed internet resources containing gene-specific experimental data and enables them to post relevant experimental results or comments. For each human gene in the system, a characterization score is posted, allowing users to track the progress of characterization over time or to identify for study uncharacterized domains in well-characterized genes. As a test of the system, a subset of 39 domains was selected for analysis and the experimental results posted to the NovelFam3000 system. For 25 human protein members of these 39 domain families, detailed sub-cellular localizations were determined. Specific observations are presented based on the analysis of the integrated information provided through the online NovelFam3000 system. CONCLUSION: Consistent experimental results between multiple members of a domain family allow for inferences of the domain's functional role. We unite bioinformatics resources and experimental data in order to accelerate the functional characterization of scarcely annotated domain families.

Animals↗

PubMed2006

Thorough validation of siRNA-induced cell death phenotypes defines new anti-apoptotic protein.

Loss-of-function by means of RNA interference in cultured human cells enables rapid pathway dissection on a genome-scale. Improved siRNA design and key validation protocols are required to eliminate falsely identified phenotypes resulting from potential off-target consequences. Here, we demonstrate a validation strategy involving several steps for verifying cell death phenotypes revealed during loss-of-function screening. First, from a set of 45 novel human genes we identified gene candidates that, when silenced, induce apoptosis in cultured HeLa cells. For those candidates, we performed more extensive validation with multiple effective siRNAs. In addition, we designed rescue experiments involving candidate genes delivered exogenously and containing silent mutations in the siRNA target regions. Rescue of the observed knockdown phenotype demonstrated an original and more stringent validation of the siRNA's selectivity and the phenotype specificity for the target gene. As a result, our data reveals an anti-apoptotic function for novel human breast adenocarcinoma marker BC-2, adding new depth to BC-2's description as a putative tumor marker involved in cancer related pathways.

Apoptosis↗

PubMed2005

Accelerated discovery of novel protein function in cultured human cells.

Experimental approaches that enable direct investigation of human protein function are necessary for comprehensive annotation of the human proteome. We introduce a cell-based platform for rapid and unbiased functional annotation of undercharacterized human proteins. Utilizing a library of antibody biomarkers, the full-length proteins are investigated by tracking phenotypic changes caused by overexpression in human cell lines. We combine reverse transfection and immunodetection by fluorescence microscopy to facilitate this procedure at high resolution. Demonstrating the advantage of this approach, new annotations are provided for two novel proteins: 1) a membrane-bound O-acyltransferase protein (C3F) that, when overexpressed, disrupts Golgi and endosome integrity due likely to an endoplasmic reticulum-Golgi transport block and 2) a tumor marker (BC-2) that prompts a redistribution of a transcriptional silencing protein (BMI1) and a mitogen-activated protein kinase mediator (Rac1) to distinct nuclear regions that undergo chromatin compaction. Our strategy is an immediate application for directly addressing those proteins whose molecular function remains unknown.

Biomarkers↗

PubMed2005

A combined approach exploring gene function based on worm-human orthology.

BACKGROUND: Many aspects of the nematode Caenorhabditis elegans biology are conserved between invertebrates and vertebrates establishing this particular organism as an excellent genetic model. Because of its small size, large populations and self-fertilization of the hermaphrodite, functional predictions carried out by genetic modifications as well as RNAi screens, can be rapidly tested. RESULTS: In order to explore the function of a set of C. elegans genes of unknown function, as well as their potential functional roles in the human genome, we performed a phylogenetic analysis to select the most probable worm orthologs. A total of 13 C. elegans genes were subjected to down-regulation via RNAi and characterization of expression profiles using GFP strains. Previously unknown distinct expression patterns were observed for four of the analyzed genes, as well as four visible RNAi phenotypes. In addition, subcellular protein over-expression profiles of the human orthologs for seven out of the thirteen genes using human cells were also analyzed. CONCLUSION: By combining a whole-organism approach using C. elegans with complementary experimental work done on human cell lines, this analysis extends currently available information on the selected set of genes.

Animals↗

PubMed2004

A novel approach for evaluating the efficiency of siRNAs on protein levels in cultured cells.

An important aim in the post-sequencing age of functional genomics is to translate gene sequences into protein functions. This shift of focus is particularly necessary for a very large number of human genes, referred to as novel genes, where we have no or very rudimentary information about their biochemical functions. Recently, a new method for investigating human gene functions using small interfering RNA molecules (siRNAs) has become available. siRNAs are powerful reagents for post-transcriptional silencing, where mRNA targeted by the siRNAs is degraded in vivo and the level of the encoded protein is reduced. However, the lack of antibodies against proteins encoded by novel genes restricts the general value of siRNAs as functional tools, as only the mRNA levels can be measured for these genes. We report a method that combines measurements of protein levels in cell culture for novel, exogenously expressed genes, with parallel measurements of the endogenous mRNA levels of the same genes. We find that this combinatorial approach correctly predicts siRNAs that efficiently reduce mRNA and protein levels in cultured cells. Furthermore, this method identifies proteins that have a slow turnover, which weakens the value of the RNA interference method as a tool for functional studies of such genes. The described method should prove to be valuable for large-scale functional studies of novel human genes.

Blotting, Western↗