Identification of differential protein interactors of lamin A and progerin

Abstract

The nuclear lamina is an interconnected meshwork of intermediate filament proteins underlying the nuclear envelope. The lamina is an important regulator of nuclear structural integrity as well as nuclear processes, including transcription, DNA replication and chromatin remodeling. The major components of the lamina are A- and B-type lamins. Mutations in lamins impair lamina functions and cause a set of highly tissue-specific diseases collectively referred to as laminopathies. The phenotypic diversity amongst laminopathies is hypothesized to be caused by mutations affecting specific protein interactions, possibly in a tissue-specific manner. Current technologies to identify interaction partners of lamin A and its mutants are hampered by the insoluble nature of lamina components. To overcome the limitations of current technologies, we developed and applied a novel, unbiased approach to identify lamin A-interacting proteins. This approach involves expression of the high-affinity OneSTrEP-tag, precipitation of lamin-protein complexes after reversible protein cross-linking and subsequent protein identification by mass spectrometry. We used this approach to identify in mouse embryonic fibroblasts and cardiac myocyte NklTAg cell lines proteins that interact with lamin A and its mutant isoform progerin, which causes the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identified a total of 313 lamina-interacting proteins, including several novel lamin A interactors, and we characterize a set of 35 proteins which preferentially interact with lamin A or progerin.

Basic concepts of epigenetics

“…Lamins directly interact with H3K9me3 and MeCP2/ Sin3A, which bind to lamin B, HP1γ and HP1α. 55,157 Lamins also adhere to lamin-associated polypeptides and, at the inner membrane, bind with emerin, Sun1 and Sun2, which anchor nesprin-3 on the outer side, where it binds to cytoskeletal actin via a Klarsicht/ANC-1/Syne homology domain. Moreover, on the outside membrane, nesprin-2 forms complexes with kinesin-1 motor protein apparatus by associating with and recruiting KLC1 to the outer nuclear membrane, which requires lamin A and C. Ultimately, the kinesin-1/nesprin-2/SUN complex spans the entire nuclear envelope in order to anchor cytoskeletal structures to nuclear lamina.…”

“…One example is the Hutchinson-Gilford progeria syndrome, which corresponds to a loss of coordinated transcriptional control of the progerin protein. 157,159 In addition, more than 200 pathogenic mutations are associated with the LMNA gene, most of which result in developmental disorders of skeletal, heart, muscle and neurological systems, such as Emery-Dreifuss muscular dystrophies, AD-limb girdle muscular dystrophy, lipodystrophy, neuropathy, cardiomyopathy, dermopathy, Atypical Werner Syndrome, hepatic stenosis, hyperpigmentation, fertility problems and accelerated aging syndromes.…”

Structures of the lamin A/C R335W and E347K mutants: Implications for dilated cardiolaminopathies

“…The substitution with a positively charged residue, as Lys, might interfere with the formation of an intra-or inter-helical salt bridge, or other forms of polar interaction. Both mutations may alter the interactions of the coiled-coil dimer, and consequently interfere with the lamina assembly; alternatively, they may affect the binding of components within the nuclear lamina, including nuclear factors, that have been proposed to interact/ associate with LMNA [40][41][42][43].…”

Mapping of protein- and chromatin-interactions at the nuclear lamina

“…This approach was combined with mild lysis, for BAF and emerin interactors, 33,44 or more stringent buffers, for lamin-LAP2β and -nesprin 2 interactions ( Table 2). 40,45 In accordance with mild solubilization, identified BAF and emerin interactors represented many proteins that also allows extraction of the total lamin A/C pool 46 ( Fig. 1) while leaving protein interactions intact.…”

“…65 Immunoprecipitations (right) with an antibody directed against lamin A/C, using mild lysis conditions (for example 0.1% NP-40, 250 mM NaCl 12 ), preferentially dissolve and precipitate nucleosoluble A-type lamins and protein interactors. 35 For a lamin A OST pull-down assay, 46 cross-linking, indicated by crosses, captures protein-protein and protein-chromatin interactions and allows solubilization of the total lamin A/C pool while preserving interactions.…”