Comparison of proteomic and metabolomic profiles of mutants of the mitochondrial respiratory chain in Caenorhabditis elegans.

Single-gene mutations that disrupt mitochondrial respiratory chain function in Caenorhabditis elegans change patterns of protein expression and metabolites. Our goal was to develop useful molecular fingerprints employing adaptable techniques to recognize mitochondrial defects in the electron transport chain. We analyzed mutations affecting complex I, complex II, or ubiquinone synthesis and discovered overarching patterns in the response of C. elegans to mitochondrial dysfunction across all of the mutations studied. These patterns are in KEGG pathways conserved from C. elegans to mammals, verifying that the nematode can serve as a model for mammalian disease. In addition, specific differences exist between mutants that may be useful in diagnosing specific mitochondrial diseases in patients.

Competition between PARP-1 and Ku70 control the decision between high-fidelity and mutagenic DNA repair.

Affinity maturation of antibodies requires a unique process of targeted mutation that allows changes to accumulate in the antibody genes while the rest of the genome is protected from off-target mutations that can be oncogenic. This targeting requires that the same deamination event be repaired either by a mutagenic or a high-fidelity pathway depending on the genomic location. We have previously shown that the BRCT domain of the DNA-damage sensor PARP-1 is required for mutagenic repair occurring in the context of IgH and IgL diversification in the chicken B cell line DT40. Here we show that immunoprecipitation of the BRCT domain of PARP-1 pulls down Ku70 and the DNA-PK complex although the BRCT domain of PARP-1 does not bind DNA, suggesting that this interaction is not DNA dependent. Through sequencing the IgL variable region in PARP-1(-/-) cells that also lack Ku70 or Lig4, we show that Ku70 or Lig4 deficiency restores GCV to PARP-1(-/-) cells and conclude that the mechanism by which PARP-1 is promoting mutagenic repair is by inhibiting high-fidelity repair which would otherwise be mediated by Ku70 and Lig4.

Integration of data for gene annotation using the BioMediator system.

Gene annotation requires integration of data from multiple sources in order to functionally classify genes. We are using BioMediator, a general purpose data-integration solution, to develop a gene annotation system to automate the process of collecting data from disparate genomic databases. Integration of annotation data from multiple sources into a single format will facilitate use of analytic tools for the proper functional classification of genes.

Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation.

We have demonstrated the use of per-methyl esterification of peptides for relative quantification of proteins between two mixtures of proteins and automated de novo sequence derivation on the same dataset. Protein mixtures for comparison were digested to peptides and resultant peptides methylated using either d0- or d3-methanol. Methyl esterification of peptides converted carboxylic acids, such as are present on the side chains of aspartic and glutamic acid as well as the carboxyl terminus, to their corresponding methyl esters. The separate d0- and d3-methylated peptide mixtures were combined and the mixture subjected to microcapillary high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Parent proteins of methylated peptides were identified by correlative database searching of peptide tandem mass spectra. Ratios of proteins in the two original mixtures could be calculated by normalization of the area under the curve for identical charge states of d0- to d3-methylated peptides. An algorithm was developed that derived, without intervention, peptide sequence de novo by comparison of tandem mass spectra of d0- and d3-peptide methyl esters.

Sequence sizes of eukaryotic enzymes.

We have shown in earlier studies that an appreciable fraction of proteins display sequence size periodicity with periods of approximately 123 aa and approximately 152 aa for eukaryotes and prokaryotes, respectively. For any firm conclusions to be made, the issue of possible bias due to an overabundance of some protein families should be addressed in more than one way. Here we present the size distributions for various sequence ensembles of eukaryotic enzymes that differ by level of data bank cleaning. The sequences were purged by applying several successive thresholds of relatedness irrespective of the sequence lengths. The previously observed preference to typical sizes is confirmed. Possible reasons for the observed excess of the typical size sequences are discussed.

Periodic recurrence of methionines: fossil of gene fusion?

As we have recently shown, approximately 20% of proteins are made of uniform size units of approximately 123 aa for eukaryotes and approximately 152 aa for prokaryotes. Such regularity may reflect certain past events in protein evolution by fusion (molecular recombination) of a spectrum of standard-size protein-coding DNA segments--the early genes. Consequently, methionines, as start residues, would mark those locations in proteins that correspond to the DNA recombination sites--the borders between the fused genes. This positional preference of the methionines may still survive as a fossil of the early protein sequence organization. In this study we address the question how methionines are distributed in modern protein sequences. This analysis of eukaryotic sequences shows that methionine residues do preferentially appear at the positions corresponding to the multiples of the unit size, as predicted.

Underlying order in protein sequence organization.

The idea of a possible standard modular structure of proteins has been known since 1929 when it was introduced by Svedberg. It still remains an idea with no quantitative confirmation of universality of such hypothetical organization. From a large collection of nonredundant protein sequences representing > 100 eukaryotic and prokaryotic species, we have obtained the protein sequence length distributions. Mere inspection of these distributions, as well as spectral analysis, shows that 15-30% of proteins, depending on species and sequence types, indeed appear to be made of sequence units with characteristic lengths of approximately 125 aa for eukaryotes and approximately 150 aa for prokaryotes. This underlying order in protein sequence organization is shown to be universal--that is, the weak regularity observed is not caused by a particular dominant species or protein group. Possible mechanisms are discussed that may be responsible for the observed regularity, including a hypothesis about the recombinational nature of such protein sequence organization.