Biology subjects

Gerace, L.

Publications and source records attributed to Gerace, L..

4 recordsLinked to original sources

bioRxiv2023

Comparative membrane proteomics reveals diverse cell regulators concentrated at the nuclear envelope

The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, largely mediated by its distinctive protein composition. We developed methods to reveal novel, low abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs to cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE.

cell biology↗

bioRxiv2022

Shared and distinctive neighborhoods of emerin and LBR revealed by proximity labeling and quantitative proteomics

Emerin and LBR are abundant transmembrane proteins of the nuclear envelope (NE) that are concentrated at the inner nuclear membrane (INM). Although both proteins interact with chromatin and nuclear lamins, they have distinctive biochemical and functional properties. Here we have deployed proximity labeling using the engineered biotin ligase TurboID (TbID) and quantitative proteomics to compare the neighborhoods of emerin and LBR in cultured mouse embryonic fibroblasts (MEFs). Our analysis revealed 232 high confidence proximity partners (HCPP) that interact selectively with emerin and/or LBR, 49 of which are shared by both. These included previously characterized NE-concentrated proteins, as well as a host of additional proteins not previously linked to emerin or LBR functions. Many of these are TM proteins of the ER and include two E3 ubiquitin ligases. Using the proximity ligation assay as an orthogonal approach, we validated the interactions described by proximity labeling for 11/12 proteins analyzed, supporting the robustness of our analysis. Overall, this work presents methodology that may be used for large-scale mapping of the landscape of the INM and reveals a group of new proteins with potential functional connections to emerin and LBR.

cell biology↗

bioRxiv2022

Lem2 is essential for cardiac development by maintaining nuclear integrity

Nuclear envelope integrity is essential for compartmentalisation of nucleus and cytoplasm. Importantly, mutations in nuclear envelope-encoding genes are the second-highest cause of familial dilated cardiomyopathy. One such nuclear envelope protein that causes cardiomyopathy in humans and affects mouse heart development is Lem2. However, its role in mechanically active tissue such as heart remains poorly understood. We generated mice in which Lem2 was specifically ablated in cardiomyocytes and carried out detailed physiological, tissue and cellular analyses. Importantly, our data showed that Lem2 was essential for cardiac development, and hearts from Lem2 cKO mice were morphologically and transcriptionally underdeveloped. Lem2 cKO hearts displayed high levels of DNA damage, nuclear rupture, and apoptosis. Crucially, we found that these defects were driven by muscle contraction as they were ameliorated by inhibiting myosin contraction and conversely were exacerbated upon myosin activation. Our data suggest that Lem2 is critical for integrity at the nascent nuclear envelope in fetal hearts, and protects the nucleus from the mechanical forces of muscle contraction. Taken together, these data provide novel insight into mechanisms underlying striated muscle diseases caused by altered nuclear envelope integrity.

cell biology↗

bioRxiv2018

Identification of new transmembrane proteins concentrated at the nuclear envelope using organellar proteomics of mesenchymal cells

The nuclear envelope (NE) is an endoplasmic reticulum (ER) subdomain that contains characteristic components dedicated to nuclear functions. These include nuclear pore complexes (NPCs) - the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) - a scaffold for NE and chromatin organization at the nuclear periphery. Since numerous human diseases associated with NE/NL proteins occur in mesenchyme-derived cells, a more comprehensive characterization of proteins concentrated at the NE in these cell types is warranted. Accordingly, we used proteomics to analyze NE and other subcellular fractions isolated from mesenchymal stem cells and from differentiated adipocytes and myocytes. We evaluated the proteomics datasets to calculate relative protein enrichment in the NE fraction, using a spectral abundance-based scoring system that accurately described most benchmark proteins. We then examined five high-scoring transmembrane proteins expressed in all three cell types that were not previously known to be enriched at the NE. Using quantitative immunofluorescence microscopy to track ectopically expressed proteins, we validated that all five of these components are substantially concentrated at the NE of multiple cell types. One (Itprip) is exposed to the outer nuclear membrane, a second (Smpd4) is enriched at the NPC, and the three others (Mfsd10, Tmx4, and Arl6ip6) are suggested to reside in the inner nuclear membrane. Considering their sequences and other features, these proteins provide new focal points for studying the functions and membrane dynamics of the NE. Our datasets should be useful for identifying additional NE-concentrated proteins, and for evaluating candidates that are identified in screening.

cell biology↗