Intact-Mass Analysis Facilitating the Identification of Large Human Heart Proteoforms.

Proteoforms, the primary effectors of biological processes, are the different forms of proteins that arise from molecular processing events such as alternative splicing and post-translational modifications. Heart diseases exhibit changes in proteoform levels, motivating the development of a deeper understanding of the heart proteoform landscape. Our recently developed two-dimensional top-down proteomics platform coupling serial size exclusion chromatography (sSEC) to reversed-phase chromatography (RPC) expanded coverage of the human heart proteome and allowed observation of high-molecular weight proteoforms. However, most of these observed proteoforms were not identified due to the difficulty in obtaining quality tandem mass spectrometry (MS2) fragmentation data for large proteoforms from complex biological mixtures on a chromatographic time scale. Herein, we sought to identify human heart proteoforms in this data set using an enhanced version of Proteoform Suite, which identifies proteoforms by intact mass alone. Specifically, we added a new feature to Proteoform Suite to determine candidate identifications for isotopically unresolved proteoforms larger than 50 kDa, enabling subsequent MS2 identification of important high-molecular weight human heart proteoforms such as lamin A (72 kDa) and trifunctional enzyme subunit α (79 kDa). With this new workflow for large proteoform identification, endogenous human cardiac myosin binding protein C (140 kDa) was identified for the first time. This study demonstrates the integration of our sSEC-RPC-MS proteomics platform with intact-mass analysis through Proteoform Suite to create a catalog of human heart proteoforms and facilitate the identification of large proteoforms in complex systems.

Lamin A molecular compression and sliding as mechanisms behind nucleoskeleton elasticity.

Lamin A is a nuclear intermediate filament protein critical for nuclear architecture and mechanics and mutated in a wide range of human diseases. Yet little is known about the molecular architecture of lamins and mechanisms of their assembly. Here we use SILAC cross-linking mass spectrometry to determine interactions within lamin dimers and between dimers in higher-order polymers. We find evidence for a compression mechanism where coiled coils in the lamin A rod can slide onto each other to contract rod length, likely driven by a wide range of electrostatic interactions with the flexible linkers between coiled coils. Similar interactions occur with unstructured regions flanking the rod domain during oligomeric assembly. Mutations linked to human disease block these interactions, suggesting that this spring-like contraction can explain in part the dynamic mechanical stretch and flexibility properties of the lamin polymer and other intermediate filament networks.

Comparative proteome analysis of a human liver cell line stably transfected with hepatitis D virus full-length cDNA.

Hepatitis D virus (HDV) is the causative agent of one of the most severe forms of virus hepatitis. HDV is a satellite virus of Hepatitis B virus (HBV) and coinfects or superinfects liver cells already infected with HBV. Investigation of HDV biology and pathogenesis has been so far impaired by the lack of an appropriate cell culture system capable of replicating and propagating the virus. This is usually partially overcome using transiently transfection systems and stably transfected cell lines. Here, we used a well-characterized human liver hepatoma cell line stably transfected with HDV cDNA (Huh7-D12) to study the changes in the host proteome induced by the expression of the virus. A 2-DE and MS approach was performed allowing the identification of 23 differentially expressed proteins when compared with the parental non-transfected Huh7 cell line. The proteomic results were validated by western blot and real-time qPCR.

Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts.

The expression of 4 pluripotency genes (Oct4, Sox2, c-Myc and Klf4) in mouse embryonic fibroblasts can reprogramme them to a pluripotent state. We have investigated the expression of these pluripotency genes when human somatic 293T cells are permeabilized and incubated in extracts of mouse embryonic stem (ES) cells. Expression of all 4 genes was induced over 1-8 h. Gene expression was associated with loss of repressive histone H3 modifications and increased recruitment of RNA polymerase II at the promoters. Lamin A/C, which is typically found only in differentiated cells, was also removed from the nuclei. When 293T cells were returned to culture after exposure to ES cell extract, the expression of the pluripotency genes continued to rise over the following 48 h of culture, suggesting that long-term reprogramming of gene expression had been induced. This provides a methodology for studying the de-differentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells to a pluripotent state without genetically altering them.

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.

Crystal structure of the human lamin A coil 2B dimer: implications for the head-to-tail association of nuclear lamins.

Nuclear intermediate filaments (IFs) are made from fibrous proteins termed lamins that assemble, in association with several transmembrane proteins of the inner nuclear membrane and an unknown number of chromatin proteins, into a filamentous scaffold called the nuclear lamina. In man, three types of lamins with significant sequence identity, i.e. lamin A/C, lamin B1 and B2, are expressed. The molecular characteristics of the filaments they form and the details of the assembly mechanism are still largely unknown. Here we report the crystal structure of the coiled-coil dimer from the second half of coil 2 from human lamin A at 2.2A resolution. Comparison to the recently solved structure of the homologous segment of human vimentin reveals a similar overall structure but a different distribution of charged residues and a different pattern of intra- and interhelical salt bridges. These features may explain, at least in part, the differences observed between the lamin and vimentin assembly pathways. Employing a modeled lamin A coil 1A dimer, we propose that the head-to-tail association of two lamin dimers involves strong electrostatic attractions of distinct clusters of negative charge located on the opposite ends of the rod domain with arginine clusters in the head domain and the first segment of the tail domain. Moreover, lamin A mutations, including several in coil 2B, have been associated with human laminopathies. Based on our data most of these mutations are unlikely to alter the structure of the dimer but may affect essential molecular interactions occurring in later stages of filament assembly and lamina formation.