GTP binding controls complex formation by the human ROCO protein MASL1

The human ROCO proteins are a family of multi-domain proteins sharing a conserved ROC-COR supra-domain. The family has four members: leu- cine-rich repeat kinase 1 (LRRK1), leucine-rich repeat kinase 2 (LRRK2), death-associated protein kinase 1 (DAPK1) and malignant fibrous histiocy- toma amplified sequences with leucine-rich tandem repeats 1 (MASL1). Previous studies of LRRK1/2 and DAPK1 have shown that the ROC (Ras of complex proteins) domain can bind and hydrolyse GTP, but the cellular consequences of this activity are still unclear. Here, the first biochemical characterization of MASL1 and the impact of GTP binding on MASL1 complex formation are reported. The results demonstrate that MASL1, similar to other ROCO proteins, can bind guanosine nucleotides via its ROC domain. Furthermore, MASL1 exists in two distinct cellular com- plexes associated with heat shock protein 60, and the formation of a low molecular weight pool of MASL1 is modulated by GTP binding. Finally, loss of GTP enhances MASL1 toxicity in cells. Taken together, these data point to a central role for the ROC/GTPase domain of MASL1 in the reg- ulation of its cellular function.

Characterization of an escherichia coli elaC deletion mutant

The elaC gene of Escherichia coli encodes a binuclear zinc phosphodiesterase (ZiPD). ZiPD homologs from various species act as 3' tRNA processing endoribonucleases, and although the homologous gene in Bacillus subtilis is essential for viability [EMBO J. 22 (2003) 4534], the physiological function of E. coli ZiPD has remained enigmatic. In order to investigate the function of E. coli ZiPD we generated and characterized an E. coli elaC deletion mutant. Surprisingly, the E. coli elaC deletion mutant was viable and had wild-type like growth properties. Micro array-based transcriptional analysis indicated expression of the E. coli elaC gene at basal levels during aerobic growth. The elaC gene deletion had no effect on the expression of genes coding for RNases or amino-acyl tRNA synthetases or any other gene among a total of > 1300 genes probed. 2D-PAGE analysis showed that the elaC mutation, likewise, had no effect on the proteome. These results strengthen doubts about the involvement of E. coli ZiPD in tRNA maturation and suggest functional diversity within the ZiPD/ElaCl protein family. In addition to these unexpected features of the E. coli elaC deletion mutant, a sequence comparison of ZiPD (ElaCl) proteins revealed specific regions for either enterobacterial or mammalian ZiPD (ElaCl) proteins. (C) 2004 Elsevier Inc. All rights reserved.

Characterization of some Actinomyces-like isolates from human clinical sources: description of Varibaculum cambriensis gen. nov., sp nov

Fifteen strains of an anaerobic, catalase-negative, gram-positive diphtheroid-shaped bacterium recovered from human sources were characterized by phenotypic and molecular chemical and molecular genetic methods. The unidentified bacterium showed some resemblance to Actinomyces species and related taxa, but biochemical testing, polyacrylamide gel electrophoresis analysis of whole-cell proteins, and amplified 16S ribosomal DNA restriction analysis indicated the strains were distinct from all currently named Actinomyces species and related taxa. Comparative 16S rRNA gene sequencing studies showed that the bacterium represents a hitherto-unknown phylogenetic line that is related to but distinct from Actinomyces, Actinobaculum, Arcanobacterium, and Mobiluncus. We propose, on the basis of phenotypic and phylogenetic evidence, that the unknown bacterium from human clinical specimens should be classified as a new genus and species, Varibaculum cambriensis gen. nov., sp. nov. The type strain of Varibaculum cambriensis sp. nov. is CCUG 44998(T) = CIP 107344(T).

Phenotypic and phylogenetic characterization of a new Corynebacterium species from dogs: description of Corynebacterium auriscanis sp. nov.

Six strains of a previously undescribed catalase-positive coryneform bacterium isolated from clinical specimens from dogs were characterized by phenotypic and molecular genetic methods. Biochemical and chemotaxonomic studies revealed that the unknown bacterium belonged to the genus Corynebacterium sensu stricto. Comparative 16S rRNA gene sequencing showed that the six strains were genealogically highly related and constitute a new subline within the genus Corynebacterium; this subline is close to but distinct from C. falsenii, C. jeikeium, and C. urealyticum. The unknown bacterium from dogs was distinguished from all currently validated Corynebacterium species by phenotypic tests including electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as a new species, Corynebacterium auriscanis. The type strain of C. auriscanis is CCUG 39938T.

Characterization of a Gemella-like organism isolated from an abscess of a rabbit: description of Gemella cunicula sp. nov.

An unknown Gram-positive, catalase-negative, ovoid-shaped bacterium isolated from the submandibular abscess of a rabbit was subjected to a polyphasic taxonomic analysis. Comparative 16S rRNA gene sequencing demonstrated the unknown coccus represents a new subline within the genus Gemella. The unknown isolate was readily distinguished from other recognized members of the genus Gemella, namely Gemella haemolysans, Gemella bergeri, Gemella morbillorum, Gemella palaticanis and Gemella sanguinis, by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on both phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium is classified in the genus Gemella as Gemella cuniculi sp. nov. The type strain is CCUG 42726T.

Characterization of Actinomyces isolates from infected root canals of teeth: description of Actinomyces radicidentis sp. nov.

Two strains of a previously undescribed Actinomyces-like bacterium were recovered in pure culture from infected root canals of teeth. Analysis by biochemical testing and polyacrylamide gel electrophoresis of whole-cell proteins indicated that the strains closely resembled each other phenotypically but were distinct from previously described Actinomyces and Arcanobacterium species. Comparative 16S rRNA gene-sequencing studies showed the bacterium to be a hitherto unknown subline within a group of Actinomyces species which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence, we propose that the unknown bacterium isolated from human clinical specimens be classified as Actinomyces radicidentis sp. nov. The type strain of Actinomyces radicidentis is CCUG 36733.

Characterization of Actinomyces isolates from samples from the human urogenital tract: description of Actinomyces urogenitalis sp. nov.

Three strains of a previously undescribed Actinomyces-like bacterium were isolated from human clinical sources (urine, urethra and vaginal secretion). Biochemical testing and PAGE analysis of whole-cell proteins indicated that the strains were phenotypically homogeneous and distinct from previously described Actinomyces and Arcanobacterium species. Comparative 16S rRNA gene sequencing studies showed the bacterium to be a hitherto unknown subline within a group of Actinomyces species which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence it is proposed that the unknown bacterium from humans be classified as Actinomyces urogenitalis sp. nov. The type strain of Actinomyces urogenitalis is CCUG 38702T (= CIP 106421T).

Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects.

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93 PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding, and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are important for the adaptation of plants to pathogenic fungi and phytophagous insects.

In silico identification and characterization of protein-ligand binding sites

Protein–ligand binding site prediction methods aim to predict, from amino acid sequence, protein–ligand interactions, putative ligands, and ligand binding site residues using either sequence information, structural information, or a combination of both. In silico characterization of protein–ligand interactions has become extremely important to help determine a protein’s functionality, as in vivo-based functional elucidation is unable to keep pace with the current growth of sequence databases. Additionally, in vitro biochemical functional elucidation is time-consuming, costly, and may not be feasible for large-scale analysis, such as drug discovery. Thus, in silico prediction of protein–ligand interactions must be utilized to aid in functional elucidation. Here, we briefly discuss protein function prediction, prediction of protein–ligand interactions, the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated EvaluatiOn (CAMEO) competitions, along with their role in shaping the field. We also discuss, in detail, our cutting-edge web-server method, FunFOLD for the structurally informed prediction of protein–ligand interactions. Furthermore, we provide a step-by-step guide on using the FunFOLD web server and FunFOLD3 downloadable application, along with some real world examples, where the FunFOLD methods have been used to aid functional elucidation.