To DNA, all information is equal.

Information storage capabilities are key in most aspects of society and the requirement for storage capacity is rapidly expanding. In principle, DNA could be a high-density medium for information storage. Church and coworkers recently demonstrated how binary data can be encoded, stored in, and retrieved from a library of oligonucleotides, increasing by several orders of magnitude the amount and density of manmade information stored in DNA to date. The technology remains in its infancy and important hurdles have yet to be overcome in order to realize its potential. However, DNA may be particularly useful as a storage-medium over long time-scales (centuries), because data-access is compatible with any large-scale DNA-sequencing and -synthesis technology.

Repo-Man coordinates chromosomal reorganization with nuclear envelope reassembly during mitotic exit.

Repo-Man targets protein phosphatase 1 γ (PP1γ) to chromatin at anaphase onset and regulates chromosome structure during mitotic exit. Here, we show that a Repo-Man:PP1 complex forms in anaphase following dephosphorylation of Repo-Man. Upon activation, the complex localizes to chromosomes and causes the dephosphorylation of histone H3 (Thr3, Ser10, and Ser28). In anaphase, Repo-Man has both catalytic and structural functions that are mediated by two separate domains. A C-terminal domain localizes Repo-Man to bulk chromatin in early anaphase. There, it targets PP1 for the dephosphorylation of histone H3 and possibly other chromosomal substrates. An N-terminal domain localizes Repo-Man to the chromosome periphery later in anaphase. There, it is responsible for the recruitment of nuclear components such as Importin ß and Nup153 in a PP1-independent manner. These observations identify Repo-Man as a key factor that coordinates chromatin remodeling and early events of nuclear envelope reformation during mitotic exit.

Structure of hibernating ribosomes studied by cryoelectron tomography in vitro and in situ.

Ribosomes arranged in pairs (100S) have been related with nutritional stress response and are believed to represent a "hibernation state." Several proteins have been identified that are associated with 100S ribosomes but their spatial organization has hitherto not been characterized. We have used cryoelectron tomography to reveal the three-dimensional configuration of 100S ribosomes isolated from starved Escherichia coli cells and we have described their mode of interaction. In situ studies with intact E. coli cells allowed us to demonstrate that 100S ribosomes do exist in vivo and represent an easily reversible state of quiescence; they readily vanish when the growth medium is replenished.

Proteomic profiling of PrP27-30-enriched preparations extracted from the brain of hamsters with experimental scrapie.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders characterized by the accumulation in the CNS of a pathological conformer (PrP(TSE)) of the host-encoded cellular prion protein (PrP(C)). PrP(TSE) has a central role in the pathogenesis of the disease but other factors are likely involved in the pathological process. In this work we employed a multi-step proteomic approach for the identification of proteins that co-purify with the protease-resistant core of PrP(TSE) (PrP27-30) extracted from brains of hamsters with experimental scrapie. We identified ferritin, calcium/calmodulin-dependent protein kinase alpha type II, apolipoprotein E, and tubulin as the major components associated with PrP27-30 but also trace amounts of actin, cofilin, Hsp90alpha, the gamma subunit of the T-complex protein 1, glyceraldehyde 3-phosphate dehydrogenase, histones, and keratins. Whereas some of these proteins (tubulin and ferritin) are known to bind PrP, other proteins (calcium/calmodulin-dependent protein kinase alpha type II, Hsp90alpha) may associate with PrP(TSE) fibrils during disease. Apolipoprotein E and actin have been previously observed in association with PrP(TSE), whereas cofilin and actin were shown to form abnormal rods in the brain of patients with Alzheimer disease. The roles of these proteins in the development of brain lesions are still unclear and further work is needed to explain their involvement in the pathogenesis of TSEs.

Improved results in proteomics by use of local and peptide-class specific false discovery rates.

BACKGROUND: Proteomic protein identification results need to be compared across laboratories and platforms, and thus a reliable method is needed to estimate false discovery rates. The target-decoy strategy is a platform-independent and thus a prime candidate for standardized reporting of data. In its current usage based on global population parameters, the method does not utilize individual peptide scores optimally. RESULTS: Here we show that proteomic analyses largely benefit from using separate treatment of peptides matching to proteins alone or in groups based on locally estimated false discovery rates. Our implementation reduces the number of false positives and simultaneously increases the number of proteins identified. Importantly, single peptide identifications achieve defined confidence and the sequence coverage of proteins is optimized. As a result, we improve the number of proteins identified in a human serum analysis by 58% without compromising identification confidence. CONCLUSION: We show that proteins can reliably be identified with a single peptide and the sequence coverage for multi-peptide proteins can be increased when using an improved estimation of false discovery rates.

Molecular and genetic analysis of condensin function in vertebrate cells.

We engineered mutants into residues of SMC2 to dissect the role of ATPase function in the condensin complex. These residues are predicted to be involved in ATP binding or hydrolysis and in the Q-loop, which is thought to act as a mediator of conformational changes induced by substrate binding. All the engineered ATPase mutations resulted in lethality when introduced into SMC2 null cells. We found that ATP binding, but not hydrolysis, is essential to allow stable condensin association with chromosomes. How SMC proteins bind and interact with DNA is still a major question. Cohesin may form a ring structure that topologically encircles DNA. We examined whether condensin behaves in an analogous way to its cohesin counterpart, and we have generated a cleavable form of biologically active condensin with PreScission protease sites engineered into the SMC2 protein. This has allowed us to demonstrate that topological integrity of the SMC2-SMC4 heterodimer is not necessary for the stability of the condensin complex in vitro or for its stable association with mitotic chromosomes. Thus, despite their similar molecular organization, condensin and cohesin exhibit fundamental differences in their structure and function.

Structural analysis of multiprotein complexes by cross-linking, mass spectrometry, and database searching.

Most protein complexes are inaccessible to high resolution structural analysis. We report the results of a combined approach of cross-linking, mass spectrometry, and bioinformatics to two human complexes containing large coiled-coil segments, the NDEL1 homodimer and the NDC80 heterotetramer. An important limitation of the cross-linking approach, so far, was the identification of cross-linked peptides from fragmentation spectra. Our novel approach overcomes the data analysis bottleneck of cross-linking and mass spectrometry. We constructed a purpose-built database to match spectra with cross-linked peptides, define a score that expresses the quality of our identification, and estimate false positive rates. We show that our analysis sheds light on critical structural parameters such as the directionality of the homodimeric coiled coil of NDEL1, the register of the heterodimeric coiled coils of the NDC80 complex, and the organization of a tetramerization region in the NDC80 complex. Our approach is especially useful to address complexes that are difficult in addressing by standard structural methods.

Proteomic analysis of human blood serum using peptide library beads.

Human serum is thought to contain key information for diagnostics of human disease. However, no single technology is currently nor might ever be available to cope with the complexity and dynamic range of the serum proteome. We here report a large-scale proteomic study of human blood serum using peptide library beads and mass spectrometry. Serum proteins are adsorbed onto polymeric beads coated with a combinatorial library composed of millions of hexameric peptide baits. Analysis of the eluates from this combinatorial library (as obtained with 3 eluants of different strength, able to release 99% of the retentate) via liquid chromatography coupled to high-resolution mass spectrometry resulted in the identification of 1559 proteins or 3869 proteins, respectively, depending on how 95% confidence was estimated. In either case, the analysis showed that ligand beads are able to capture a large number of proteins in a single operation. The ligand bead bound fraction appeared to have a lower dynamic range when compared to the starting material, due to a "normalization" of the protein concentrations in the original mixture. We find that extensive information on the protein composition of complex samples such as serum can be obtained using ligand beads and that these beads enrich the proteomic tool box.

RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3.

Methylation of histones has been regarded as a stable modification defining the epigenetic program of the cell, which regulates chromatin structure and transcription. However, the recent discovery of histone demethylases has challenged the stable nature of histone methylation. Here we demonstrate that the JARID1 proteins RBP2, PLU1, and SMCX are histone demethylases specific for di- and trimethylated histone 3 lysine 4 (H3K4). Consistent with a role for the JARID1 Drosophila homolog Lid in regulating expression of homeotic genes during development, we show that RBP2 is displaced from Hox genes during embryonic stem (ES) cell differentiation correlating with an increase of their H3K4me3 levels and expression. Furthermore, we show that mutation or RNAi depletion of the C. elegans JARID1 homolog rbr-2 leads to increased levels of H3K4me3 during larval development and defects in vulva formation. Taken together, these results suggest that H3K4me3/me2 demethylation regulated by the JARID1 family plays an important role during development.

Exploring the hidden human urinary proteome via ligand library beads.

The human urinary proteome has been reassessed and re-evaluated via a novel concentration/equalization technique, exploiting beads coated with hexameric peptide ligand libraries. These beads act by capturing the whole protein spectra contained in the sample, by drastically reducing the level of the most abundant species, while strongly concentrating the more dilute and rare ones. In a control urine sample, 134 unique proteins could be identified. The first bead eluate (in thiourea, urea, and CHAPS) permitted the identification of 317 gene products, whereas the second eluate (in 9 M urea, pH 3.8) allowed the identification of another 95 unique proteins. By eliminating redundancies, a total of 383 unique gene products could be identified in human urines. This represents a major increment as compared to data reported in recent literature. By comparing our data with those reported to the present, an additional 251 proteins could be added to the list, thus bringing the total unique gene products so far identified in human urines to ca. 800 species.