Purification of Lamins and Soluble Fragments of NETs.

Lamins and associated nuclear envelope transmembrane proteins (NETs) present unique problems for biochemical studies. Lamins form insoluble intermediate filament networks, associate with chromatin, and are also connected via specific NETs to the cytoskeleton, thus further complicating their isolation and purification from mammalian cells. Adding to this complexity, NETs at the inner nuclear membrane function in three distinct environments: (a) their nucleoplasmic domain(s) can bind lamins, chromatin, and transcriptional regulators; (b) they possess one or more integral transmembrane domains; and (c) their lumenal domain(s) function in the unique reducing environment of the nuclear envelope/ER lumen. This chapter describes strategic considerations and protocols to facilitate biochemical studies of lamins and NET proteins in vitro. Studying these proteins in vitro typically involves first expressing specific polypeptide fragments in bacteria and optimizing conditions to purify each fragment. We describe parameters for choosing specific fragments and designing purification strategies and provide detailed purification protocols. Biochemical studies can provide fundamental knowledge including binding strengths and the molecular consequences of disease-causing mutations that will be essential to understand nuclear envelope-genome interactions and nuclear envelope linked disease mechanisms.

Comparative Label-Free Mass Spectrometric Analysis of Mildly versus Severely Affected mdx Mouse Skeletal Muscles Identifies Annexin, Lamin, and Vimentin as Universal Dystrophic Markers.

The primary deficiency in the membrane cytoskeletal protein dystrophin results in complex changes in dystrophic muscles. In order to compare the degree of secondary alterations in differently affected subtypes of skeletal muscles, we have conducted a global analysis of proteome-wide changes in various dystrophin-deficient muscles. In contrast to the highly degenerative mdx diaphragm muscle, which showed considerable alterations in 35 distinct proteins, the spectrum of mildly to moderately dystrophic skeletal muscles, including interosseus, flexor digitorum brevis, soleus, and extensor digitorum longus muscle, exhibited a smaller number of changed proteins. Compensatory mechanisms and/or cellular variances may be responsible for differing secondary changes in individual mdx muscles. Label-free mass spectrometry established altered expression levels for diaphragm proteins associated with contraction, energy metabolism, the cytoskeleton, the extracellular matrix and the cellular stress response. Comparative immunoblotting verified the differences in the degree of secondary changes in dystrophin-deficient muscles and showed that the up-regulation of molecular chaperones, the compensatory increase in proteins of the intermediate filaments, the fibrosis-related increase in collagen levels and the pathophysiological decrease in calcium binding proteins is more pronounced in mdx diaphragm as compared to the less severely affected mdx leg muscles. Annexin, lamin, and vimentin were identified as universal dystrophic markers.

Identification of new in vivo phosphosites on lamin Dm - the evidence of heterogeneity of phosphorylation sites in different Drosophila tissues.

Changes in the nuclear structure and function during the cell cycle are thought to be correlated with lamins phosphorylation. Here, we report the identification of new in vivo phosphorylation sites on Drosophila melanogater lamin Dm using immunoisolation and mass spectrometry with collision-induced peptide fragmentation (Electrospray-Linear Trap Quadrupole- Fourier Transform Ion Cyclotron Resonance MS/MS). We identified S19 and confirmed previously suggested S595 as phosphorylated amino acid residues on embryonic lamin Dm. We also found that T597 is phosphorylated in vivo in cultured Kc cells while S595 in embryos, which suggests that different neighboring phosphoacceptors may be modified within the same region. We demonstrate also that Drosophila melanogaster lamin Dm in very early (syncytial) embryos is almost completely dispersed through the entire embryo. Only fraction of lamin Dm is associated with nuclei and nuclear envelopes. In later stages, due to the synchronization of mitosis, lamin Dm may be both nuclear and cytoplasmic in the same embryo. Our results provide a new and essential data for better understanding of the lamin phosporylation in development and cell cycle regulation in Drosophila.

Lamins of ocular lens epithelial cells.

Experiments were performed to characterize a prominent nuclear matrix (NM) protein isolated from tissue cultured mouse lens epithelial cells. This NM protein was separated by SDS-PAGE and the stained gel band was analyzed by mass spectroscopy. Blast analysis of the amino acid sequence derived by mass spectroscopy revealed the presence of Lamin C in the NM of the mouse lens epithelial cells. We also examined nuclear proteins of adult and fetal human lenses. Data collected from these experiments showed the presence of Lamin C in both adult and fetal lens cells. However fetal lens cells only show Lamin C dimers, whereas adult human lens contained dimers, monomers and degraded Lamin C. Early and late passaged tissue cultured mouse lens epithelial cells also contained Lamin C in the nucleus with a preponderance of the dimer in the early passaged cells. The biological significance of the presence of dimers in human fetal lens cells and early passaged mouse lens cells is not known. However, it could suggest an enhanced docking capability of Lamin C dimers for other physiologically important nuclear proteins.

Proteome alterations in human host cells infected with coxsackievirus B3.

Coxsackievirus B3 (CVB3) is a common factor in human myocarditis. The interplay between host factors and virus components is crucial for the fate of the infected cells. Despite that, host protein responses, which characterize CVB3-induced diseases, have not yet been determined in detail. To investigate the nature of modified protein patterns in infected human cells compared with uninfected cells, two-dimensional gel electrophoresis in combination with matrix-assisted laser desorption/ionization-mass spectrometry were used. The regulated proteins, e.g. nucleophosmin (nucleolar protein B23), lamin, the RNA-binding protein UNR and the p38 mitogen-activated protein kinase, were sorted according to their functional groups and interpreted in the context of the myocarditis process.