NovelFam3000--uncharacterized human protein domains conserved across model organisms

Abstract

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.

Figure 1

Screenshots of the NovelFam3000 interface. (a) The protein domain page displays protein members across model organisms for a given protein domain family. Detailed information about each gene is available via various hyperlinks including news, resource links, experimental evidence, and comments. Each link takes the user to a separate page with available link-specific information and the option to submit new data and comments. (b) The resource link displays hyperlinks to a set of bioinformatics databases containing information for a specific domain family member. For this human example, resources are divided into genomic resources, molecular interactions, and protein networks and resources. The user has the option to submit new resource links to the system.

Figure 2

Immunolocalization of protein domain family PF04427 members. Human FLAG-tagged recombinant proteins detected with FLAG-specific antibodies in human PLC/PRF/5 cells (a: BRIX_HUMAN; e: IMP4). Fly His-tagged recombinant proteins detected with His-specific antibodies in fly cells (i: CG11920: l: CG32253). Fly GFP-tagged recombinant proteins detected in human HEK293 cells (n: CG32253; r: CG6712). Fibrillarin staining of nucleoli (b, f, o, s); composite images between proceeding stains (c, g, p, t); DAPI staining of nuclei (d, h, k, m, q, u).

Figure 3

Immunolocalization of protein domain family PF03114 members. FLAG-tagged recombinant proteins detected with FLAG-specific antibodies (a: SH3GL1; c: SH3BP1); DAPI staining of nuclei (b, d). Both proteins show similar vesicular localization patterns. The pattern is distinct from those obtained with multiple cellular markers for vesicle structures.

Figure 4

Diverse localization patterns observed by immunolocalization of FLAG-tagged recombinant proteins detected with FLAG-specific antibodies. NP_057480 (HSPC129) of protein domain family PF03031 localizes to the nuclear rim (a), DULLARD is found in the ER (c) as confirmed by calnexin counterstaining (d, e), and CTDSPL is present in mitochondria (g) as confirmed by staining with MitoTracker Red CMX dye (h, i). DAPI staining for nuclei (b, f, k).

Figure 5

Novel immunolocalization pattern of FLAG-tagged recombinant protein detected with FLAG-specific antibodies (a). DAPI staining for nuclei (b). NP_055268 (CHMP2A, BC-2) of protein family PF03357 forms distinct cytoplasmic structures in PLC/PRF/5 cells.