Luitzen de Jong

Binding of matrix attachment regions to lamin B1

Eukaryotic chromatin is organized into topologically constrained loops that are attached to the nuclear matrix. The regions of DNA that interact with the matrix are called matrix attachment regions (MARs). We studied the spatial distribution of MAR-binding sites in the nuclear matrix from rat liver cells, following a combined biochemical and ultrastructural approach. We found that MAR-binding sites are distributed equally over the internal fibrogranular network and the peripheral nuclear lamina. Internal and peripheral binding sites have similar binding characteristics: both sets of binding sites show specific and saturable binding of MARs from different organisms. By means of a DNA-binding protein blot assay and in vitro binding studies, we identified lamin B1 as a MAR-binding protein, which provides evidence for a specific interaction of DNA with the nuclear lamina.

Proteomic Analysis of Candida albicans Cell Walls Reveals Covalently Bound Carbohydrate-Active Enzymes and Adhesins

ABSTRACT Covalently linked cell wall proteins (CWPs) of the dimorphic fungus Candida albicans are implicated in virulence. We have carried out a comprehensive proteomic analysis of the covalently linked CWPs in exponential-phase yeast cells. Proteins were liberated from sodium dodecyl sulfate (SDS)-extracted cell walls and analyzed using immunological and advanced protein sequencing (liquid chromatography-tandem mass spectrometry [LC/MS/MS]) methods. HF-pyridine and NaOH were used to chemically release glycosylphosphatidylinositol-dependent proteins (GPI proteins) and mild alkali-sensitive proteins, respectively. In addition, to release both classes of CWPs simultaneously, cell walls were digested enzymatically with a recombinant β-1,3-glucanase. Using LC/MS/MS, we identified 14 proteins, of which only 1 protein, Cht2p, has been previously identified in cell wall extracts by using protein sequencing methods. The 14 identified CWPs include 12 GPI proteins and 2 mild alkali-sensitive proteins. Nonsecretory proteins were absent in our cell wall preparations. The proteins identified included several functional categories: (i) five CWPs are predicted carbohydrate-active enzymes (Cht2p, Crh11p, Pga4p, Phr1p, and Scw1p); (ii) Als1p and Als4p are believed to be adhesion proteins. In addition, Pga24p shows similarity to the flocculins of bakers yeast. (iii) Sod4p/Pga2p is a putative superoxide dismutase and is possibly involved in counteracting host defense reactions. The precise roles of the other CWPs (Ecm33.3p, Pir1p, Pga29p, Rbt5p, and Ssr1p) are unknown. These results indicate that a substantial number of the covalently linked CWPs of C. albicans are actively involved in cell wall remodeling and expansion and in host-pathogen interactions.

Chemical cross-linking and mass spectrometry for protein structural modeling

The growth of gene and protein sequence information is currently so rapid that three-dimensional structural information is lacking for the overwhelming majority of known proteins. In this review, efforts towards rapid and sensitive methods for protein structural characterization are described, complementing existing technologies. Based on chemical cross-linking and offering the analytical speed and sensitivity of mass spectrometry these methodologies are thought to contribute valuable tools towards future high throughput protein structure elucidation.

A structure for the yeast prohibitin complex: Structure prediction and evidence from chemical crosslinking and mass spectrometry

The mitochondrial prohibitin complex consists of two subunits (PHB1 of 32 kD and PHB2 of 34 kD), assembled into a membrane‐associated supercomplex of approximately 1 MD. A chaperone‐like function in holding and assembling newly synthesized mitochondrial polypeptide chains has been proposed. To further elucidate the function of this complex, structural information is necessary. In this study we use chemical crosslinking, connecting lysine side chains, which are well scattered along the sequence. Crosslinked peptides from protease digested prohibitin complexes were identified with mass spectrometry. From these results, spatial restraints for possible protein conformation were obtained. Many interaction sites between PHB1 and PHB2 were found, whereas no homodimeric interactions were observed. Secondary and tertiary structural predictions were made using several algorithms and the models best fitting the spatial restraints were selected for further evaluation. From the structure predictions and the crosslink data we derived a structural building block of one PHB1 and one PHB2 subunit, strongly intertwined along most of their length. The size of the complex implies that approximately 14 of these building blocks are present. Each unit contains a putative transmembrane helix in PHB2. Taken together with the unit building block we postulate a circular palisade‐like arrangement of the building blocks projecting into the intermembrane space.

hnRNP proteins and B23 are the major proteins of the internal nuclear matrix of HeLa S3 cells

The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina‐pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa S3 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high‐resolution two‐dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA.

PML-Containing Nuclear Bodies: Their Spatial Distribution in Relation to Other Nuclear Components

The PML protein is a human growth suppressor concentrated in 10 to 20 nuclear bodies per nucleus (PML bodies). Disruption of the PML gene has been shown to be related to acute promyelocytic leukaemia (APL). To obtain information about the function of PML bodies we have investigated the 3D‐distribution of PML bodies in the nucleus of T24 cells and compared it with the spatial distribution of a variety of other nuclear components, using fluorescence dual‐labeling immunocytochemistry and confocal microscopy. Results show that PML bodies are not enriched in nascent RNA, the splicing component U2‐snRNP, or transcription factors (glucocorticoid receptor, TFIIH, and E2F). These results show that PML bodies are not prominent sites of RNA synthesis or RNA splicing. We found that a large fraction of PML bodies (50 to 80%) is closely associated with DNA replication domains during exclusively middle‐late S‐phase. Furthermore, in most cells that we analysed we found at least one PML body was tightly associated with a coiled body. In the APL cell line NB4, the PML gene is fused with the RARα gene due to a chromosomal rearrangement. PML bodies have disappeared and the PML antigen, i.e., PML and the PML‐RAR fusion protein, is dispersed in a punctated pattern throughout the nucleoplasm. We showed that in NB4 cells the sites that are rich in PML antigen significantly colocalize with sites at which nascent RNA accumulates. This suggests that, in contrast to non‐APL cells, in NB4 cells the PML antigen is associated with sites of transcription. The implications of these findings for the function of PML bodies are consistent with the idea that PML bodies are associated with specific genomic loci.

Nuclear Domains and the Nuclear Matrix

This overview describes the spatial distribution of several enzymatic machineries and functions in the interphase nucleus. Three general observations can be made. First, many components of the different nuclear machineries are distributed in the nucleus in a characteristic way for each component. They are often found concentrated in specific domains. Second, nuclear machineries for the synthesis and processing of RNA and DNA are associated with an insoluble nuclear structure, called nuclear matrix. Evidently, handling of DNA and RNA is done by immobilized enzyme systems. Finally, the nucleus seems to be divided in two major compartments. One is occupied by compact chromosomes, the other compartment is the space between the chromosomes. In the latter, transcription takes place at the surface of chromosomal domains and it houses the splicing machinery. The relevance of nuclear organization for efficient gene expression is discussed.

A new crosslinker for mass spectrometric analysis of the quaternary structure of protein complexes.

Mass spectrometric structural analysis of crosslinked peptides is a powerful method to elucidate the spatial arrangement of polypeptides in protein complexes. Our aim is to develop bifunctional crosslinkers that, after crosslinking protein complexes followed by proteolytic digestion, give rise to crosslinked peptides that can be readily tracked down by mass spectrometry. To this end we synthesized the crosslinker N-benzyliminodiacetoyloxysuccinimid (BID), which yields stable benzyl cation marker ions upon low-energy collisioninduced dissociation (CID) tandem mass spectrometry. Sensitive detection of the marker ion upon low-energy CID is demonstrated with different BID-crosslinked peptide preparations. With BID it becomes possible to retrieve crosslinked and crosslinker-adducted peptides, without the necessity of purifying crosslinked peptides prior to identification. The basic design of this crosslinker can be varied upon, in order to meet specific crosslinking needs.

Selective Enrichment of Azide-Containing Peptides from Complex Mixtures

A general method is described to sequester peptides containing azides from complex peptide mixtures, aimed at facilitating mass spectrometric analysis to study different aspects of proteome dynamics. The enrichment method is based on covalent capture of azide-containing peptides by the azide-reactive cyclooctyne (ARCO) resin and is demonstrated for two different applications. Enrichment of peptides derived from cytochrome c treated with the azide-containing cross-linker bis(succinimidyl)-3-azidomethyl glutarate (BAMG) shows several cross-link containing peptides. Sequestration of peptides derived from an Escherichia coli proteome, pulse labeled with the bio-orthogonal amino acid azidohomoalanine as substitute for methionine, allows identification of numerous newly synthesized proteins. Furthermore, the method is found to be very specific, as after enrichment over 87% of all peptides contain (modified) azidohomoalanine.

Nuclear neighbours: the spatial and functional organization of genes and nuclear domains.

It is becoming clear that the cell nucleus is not only organized in domains but that these domains are also organized relative to each other and to the genome. Specific nuclear domains, enriched in different proteins and RNAs, are often found next to each other and next to specific gene loci. Several lines of investigation suggest that nuclear domains are involved in facilitating or regulating gene expression. The emerging view is that the spatial relationship between different domains and genes on different chromosomes, as found in the nucleolus, is a common organizational principle in the nucleus, to allow an efficient and controlled synthesis and processing of a range of gene transcripts. J. Cell. Biochem. 70:159–171.