RESUMO
In order to circumvent limitations of sequence based methods in the process of making functional predictions for proteins, we have developed a methodology that uses a sequence-to-structure-to-function paradigm. First, an approximate three-dimensional structure is predicted. Then, a three-dimensional descriptor of the functional site, termed a Fuzzy Functional Form, or FFF, is used to screen the structure for the presence of the functional site of interest (Fetrow et al., 1998; Fetrow and Skolnick, 1998). Previously, a disulfide oxidoreductase FFF was developed and applied to predicted structures obtained from a small structural database. Here, using a substantially larger structural database, we expand the analysis of the disulfide oxidoreductase FFF to the B. subtilis genome. To ascertain the performance of the FFF, its results are compared to those obtained using both the sequence alignment method BLAST and three local sequence motif databases: PRINTS, Prosite, and Blocks. The FFF method is then compared in detail to Blocks and it is shown that the FFF is more flexible and sensitive in finding a specific function in a set of unknown proteins. In addition, the estimated false positive rate of function prediction is significantly lower using the FFF structural motif, rather than the standard sequence motif methods. We also present a second FFF and describe a specific example of the results of its whole-genome application to D. melanogaster using a newer threading algorithm. Our results from all of these studies indicate that the addition of three-dimensional structural information adds significant value in the prediction of biochemical function of genomic sequences.
Assuntos
Proteínas/química , Proteínas/fisiologia , Algoritmos , Sequência de Aminoácidos , Animais , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Genoma , Genoma Bacteriano , Humanos , Proteínas de Insetos/genética , Proteínas de Insetos/fisiologia , Dados de Sequência Molecular , Proteína Dissulfeto Redutase (Glutationa)/química , Proteína Dissulfeto Redutase (Glutationa)/fisiologia , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/fisiologia , Proteínas/genética , Relação Estrutura-AtividadeRESUMO
Serum from an infertile male with high-titer anti-sperm antibodies was used to identify a novel human sperm antigen by screening of a testis expression library. The clone, initially designated Repro-SA-1 (HUGO-approved symbol SPAG6), was found to encode a sequence highly enriched in testis. The deduced amino acid sequence of the full-length cDNA revealed striking homology to the product of the Chlamydomonas reinhardtii PF16 locus, which encodes a protein localized to the central pair of the flagellar axoneme. The human gene encodes 1.8- and 2.8-kb mRNAs highly expressed in testis but not in prostate, ovary, spleen, thymus, small intestine, colon, peripheral blood leukocytes, heart, brain, placenta, liver, muscle, kidney, and pancreas. The gene was mapped to chromosome 10p11.2-p12. Antibodies raised against SPAG6 sequences localized the protein to the tails of permeabilized human sperm. Both the Chlamydomonas protein and SPAG6 contain eight contiguous armadillo repeats, which place them in a family of proteins known to mediate protein-protein interactions. The cloning of the human homologue of the Chlamydomonas PF16 locus provides a new avenue to explore the role of the axoneme central pair in human sperm function.
Assuntos
Proteínas de Algas , Proteínas dos Microtúbulos/genética , Espermatozoides/química , Espermatozoides/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/sangue , Antígenos/química , Antígenos/genética , Antígenos/imunologia , Chlamydomonas/genética , Chlamydomonas reinhardtii/genética , Células Clonais , Clonagem Molecular , Sequência Conservada , DNA/biossíntese , Bases de Dados Factuais , Expressão Gênica , Biblioteca Gênica , Humanos , Soros Imunes , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Infertilidade Masculina/sangue , Infertilidade Masculina/genética , Infertilidade Masculina/imunologia , Masculino , Proteínas dos Microtúbulos/química , Proteínas dos Microtúbulos/imunologia , Dados de Sequência Molecular , Filogenia , RNA/análise , Proteínas Recombinantes/imunologia , Homologia de Sequência de Aminoácidos , Testículo/química , Distribuição TecidualRESUMO
I-POU, a POU domain nuclear protein that lacks two conserved basic amino acids of the POU homeodomain is coexpressed in the developing Drosophila nervous system with a second POU domain transcription factor, Cf1-a. I-POU does not bind to DNA but forms a POU domain-mediated, high affinity heterodimer with Cf1-a, inhibiting its ability to bind and activate the dopa decarboxylase gene. The I-POU/Cf1-a dimerization interface encompasses only the N-terminal basic region and helices 1 and 2 of the POU homeodomains with precise amino acid and alpha-helical requirements. twin of I-POU, an alternatively spliced transcript of the I-POU gene, encodes a protein containing the two basic amino acid residues absent in I-POU. Twin of I-POU is incapable of dimerizing with Cf1-a, but can act as a positive transcription factor on targets distinct from those regulated by Cf1-a. These findings suggest that the I-POU genomic locus simultaneously generates both a specific activator and inhibitor of gene transcription, capable of modulating two distinct regulatory programs during neural development.
