Systematic Characterization of Nuclear Proteome during Apoptosis

Abstract

Identification and characterization of the nuclear proteome is important for detailed understanding of multiple signaling events in eukaryotic cells. Toward this goal, we extensively characterized the nuclear proteome of human T leukemia cells by sequential extraction of nuclear proteins with different physicochemical properties using three buffer conditions. This large scale proteomic study also tested the feasibility and technical challenges associated with stable isotope labeling by amino acids in cell culture (SILAC) to uncover quantitative changes during apoptosis. Analyzing proteins from three nuclear fractions extracted from naïve and apoptotic cells generated 780,530 MS/MS spectra that were used for database searching using the SEQUEST algorithm. This analysis resulted in the identification and quantification of 1,174 putative nuclear proteins. A number of known nuclear proteins involved in apoptosis as well as novel proteins not known to be part of the nuclear apoptotic machinery were identified and quantified. Consistent with SILACbased quantifications, immunofluorescence staining of nucleus, mitochondria, and some associated proteins from both organelles revealed a dynamic recruitment of mitochondria into nuclear invaginations during apoptosis. Molecular & Cellular Proteomics 5:1131-1145, 2006.Comprehensive understanding of the biology of the nucleus will require complete identification of the proteome of this important organelle. The nucleus of the cell is where a number of key enzymatic processes occur, and most of these processes are critical for cellular homeostasis: DNA synthesis, DNA replication and repair, transcription, higher order chromatin organization, and gene and chromosomal silencing (1-8). A comprehensive list of proteins that reside in the nucleus, as well as the proteins that shuttle between multiple subcellular compartments and nucleus, has not been completed to date (9 -11). In addition, different classes of proteins are sequestered in the nucleus based on their affinity to DNA, nuclear matrix, nuclear membrane, nucleolus, specialized nuclear bodies such as speckles and PIKA (polymorphic interphase karyosomal association), or Cajal bodies (coiled bodies) (12-15). Thus, large scale identification of nuclear proteins from human cells and characterization of their associated physicochemical properties will likely provide insights into the biology of the nucleus.We have been interested in identifying proteins that control apoptosis in the nucleus. Apoptosis or programmed cell death is a process essential for the development and maintenance of cellular homeostasis of higher eukaryotes (16). One of the hallmarks of apoptosis is rapid chromatin condensation and DNA fragmentation (17). The exact mechanisms that control rapid DNA fragmentation and chromatin condensation in the nucleus are not fully understood, although some of the proteins that are crucial for these events have been identified (18 -25). For example, the caspase-activated DNase (CAD/DFF40) (18, 19), lamin protease (Caspase 6...

Quantitative proteome analysis of cisplatin‐induced apoptotic Jurkat T cells by stable isotope labeling with amino acids in cell culture, SDS‐PAGE, and LC‐MALDI‐TOF/TOF MS

“…During analysis by MS, relative peak intensities between unlabeled and labeled peptides are quantitative, enabling to evaluate the effects of specific treatment on a large number of proteins within a single experiment. Apoptosis was already studied by SILAC-based quantitative proteome analysis of CD95 (Fas/Apo-1)-induced apoptosis in combination with 2-DE and MALDI-MS of total cell lysates [20] and by SDS-PAGE and m-LC-MS/MS of mitochondrial and nuclear proteins [21,22].…”

Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT)

“…These methods have successfully elucidated complex processes such as nuclear protein dynamics (7,9), responses to cytokines (10), and mechanisms of neurodegenerative diseases (11,12), as well as promoting biomarker discovery (13)(14)(15). More recently, approaches focusing on posttranslational modifications have emerged to specifically explore the ''phosphoproteome'' (16)(17)(18) and ''glycoproteome'' (19)(20)(21)(22) of a cell.…”

Mapping the Interactome of a Major Mammalian Endoplasmic Reticulum Heat Shock Protein 90

“…Tryptic peptides from each of the gel slices were analyzed using an LTQ linear ion trap mass spectrometer (Thermo Finnigan, San Jose, CA), as described previously [27]. The solvent gradient of HPLC was linear from 100% solvent A (5% acetonitrile, 0.4% acetic acid, and 0.005% heptafluorobutyric acid) to 80% solvent B (100% acetonitrile, 0.4% acetic acid, and 0.005% heptafluorobutyric acid) for 78 minutes, with a 20-65- minute acquisition.…”