In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice.

Hutchinson-Gilford progeria syndrome (HGPS or progeria) is typically caused by a dominant-negative C•G-to-T•A mutation (c.1824 C>T; p.G608G) in LMNA, the gene that encodes nuclear lamin A. This mutation causes RNA mis-splicing that produces progerin, a toxic protein that induces rapid ageing and shortens the lifespan of children with progeria to approximately 14 years1-4. Adenine base editors (ABEs) convert targeted A•T base pairs to G•C base pairs with minimal by-products and without requiring double-strand DNA breaks or donor DNA templates5,6. Here we describe the use of an ABE to directly correct the pathogenic HGPS mutation in cultured fibroblasts derived from children with progeria and in a mouse model of HGPS. Lentiviral delivery of the ABE to fibroblasts from children with HGPS resulted in 87-91% correction of the pathogenic allele, mitigation of RNA mis-splicing, reduced levels of progerin and correction of nuclear abnormalities. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. In transgenic mice that are homozygous for the human LMNA c.1824 C>T allele, a single retro-orbital injection of adeno-associated virus 9 (AAV9) encoding the ABE resulted in substantial, durable correction of the pathogenic mutation (around 20-60% across various organs six months after injection), restoration of normal RNA splicing and reduction of progerin protein levels. In vivo base editing rescued the vascular pathology of the mice, preserving vascular smooth muscle cell counts and preventing adventitial fibrosis. A single injection of ABE-expressing AAV9 at postnatal day 14 improved vitality and greatly extended the median lifespan of the mice from 215 to 510 days. These findings demonstrate the potential of in vivo base editing as a possible treatment for HGPS and other genetic diseases by directly correcting their root cause.

Lamin A K97E leads to NF-κB-mediated dysfunction of inflammatory responses in dilated cardiomyopathy.

BACKGROUND INFORMATION: Lamins are type V intermediate filament proteins underlying the inner nuclear membrane which provide structural rigidity to the nucleus, tether the chromosomes, maintain nuclear homeostasis, and remain dynamically associated with developmentally regulated regions of the genome. A large number of mutations particularly in the LMNA gene encoding lamin A/C results in a wide array of human diseases, collectively termed as laminopathies. Dilated Cardiomyopathy (DCM) is one such laminopathic cardiovascular disease which is associated with systolic dysfunction of left or both ventricles leading to cardiac arrhythmia which ultimately culminates into myocardial infarction. RESULTS: In this work, we have unraveled the epigenetic landscape to address the regulation of gene expression in mouse myoblast cell line in the context of the missense mutation LMNA 289A

Cyclin-dependent kinase 1 depolymerizes nuclear lamin filaments by disrupting the head-to-tail interaction of the lamin central rod domain.

Nuclear lamins maintain the nuclear envelope structure by forming long linear filaments via two alternating molecular arrangements of coiled-coil dimers, known as A11 and A22 binding modes. The A11 binding mode is characterized by the antiparallel interactions between coil 1b domains, whereas the A22 binding mode is facilitated by interactions between the coil 2 domains of lamin. The junction between A11- and A22-interacting dimers in the lamin tetramer produces another parallel head-tail interaction between coil 1a and the C-terminal region of coil 2, called the ACN interaction. During mitosis, phosphorylation in the lamin N-terminal head region by the cyclin-dependent kinase (CDK) complex triggers depolymerization of lamin filaments, but the associated mechanisms remain unknown at the molecular level. In this study, we revealed using the purified proteins that phosphorylation by the CDK1 complex promotes disassembly of lamin filaments by directly abolishing the ACN interaction between coil 1a and the C-terminal portion of coil 2. We further observed that this interaction was disrupted as a result of alteration of the ionic interactions between coil 1a and coil 2. Combined with molecular modeling, we propose a mechanism for CDK1-dependent disassembly of the lamin filaments. Our results will help to elucidate the cell cycle-dependent regulation of nuclear morphology at the molecular level.

Site specificity determinants for prelamin A cleavage by the zinc metalloprotease ZMPSTE24.

The integral membrane zinc metalloprotease ZMPSTE24 is important for human health and longevity. ZMPSTE24 performs a key proteolytic step in maturation of prelamin A, the farnesylated precursor of the nuclear scaffold protein lamin A. Mutations in the genes encoding either prelamin A or ZMPSTE24 that prevent cleavage cause the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS) and related progeroid disorders. ZMPSTE24 has a novel structure, with seven transmembrane spans that form a large water-filled membrane chamber whose catalytic site faces the chamber interior. Prelamin A is the only known mammalian substrate for ZMPSTE24; however, the basis of this specificity remains unclear. To define the sequence requirements for ZMPSTE24 cleavage, we mutagenized the eight residues flanking the prelamin A scissile bond (TRSY↓LLGN) to all other 19 amino acids, creating a library of 152 variants. We also replaced these eight residues with sequences derived from putative ZMPSTE24 cleavage sites from amphibian, bird, and fish prelamin A. Cleavage of prelamin A variants was assessed using an in vivo yeast assay that provides a sensitive measure of ZMPSTE24 processing efficiency. We found that residues on the C-terminal side of the cleavage site are most sensitive to changes. Consistent with other zinc metalloproteases, including thermolysin, ZMPSTE24 preferred hydrophobic residues at the P1' position (Leu647), but in addition, showed a similar, albeit muted, pattern at P2'. Our findings begin to define a consensus sequence for ZMPSTE24 that helps to clarify how this physiologically important protease functions and may ultimately lead to identifying additional substrates.

Structural basis for the interaction between unfarnesylated progerin and the Ig-like domain of lamin A/C in premature aging disorders.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by C-terminally truncated lamin A, termed as the pre-progerin product. Progerin is a C-terminally farnesylated protein derived from pre-progerin, which causes nuclear deformation at the inner-nuclear membrane. As an alternative or additional mechanism, a farnesylation-independent abnormal interaction between the C-terminus of progerin and Ig-like domain has been proposed. However, the molecular mechanism underlying the role of unfarnesylated C-terminus of pre-progerin in HGPS remains largely unknown. In this study, we determined the crystal structures of C-terminal peptide of progerin and Ig-like domain of lamin A/C. Results showed that the C-terminal cysteine residue of progerin forms a disulfide bond with the only cysteine residue of the Ig-like domain. This finding suggested that unfarnesylated progerin can form a disulfide bond with the Ig-like domain in the lamin meshwork. The Alphafold2-assisted docking structure showed that disulfide bond formation was promoted by a weak interaction between the groove of Ig-like domain and the unfarnesylated C-terminal tail region of progerin. Our results provide molecular insights into the normal aging process as well as premature aging of humans.

Concentric organization of A- and B-type lamins predicts their distinct roles in the spatial organization and stability of the nuclear lamina.

The nuclear lamina is an intermediate filament meshwork adjacent to the inner nuclear membrane (INM) that plays a critical role in maintaining nuclear shape and regulating gene expression through chromatin interactions. Studies have demonstrated that A- and B-type lamins, the filamentous proteins that make up the nuclear lamina, form independent but interacting networks. However, whether these lamin subtypes exhibit a distinct spatial organization or whether their organization has any functional consequences is unknown. Using stochastic optical reconstruction microscopy (STORM) our studies reveal that lamin B1 and lamin A/C form concentric but overlapping networks, with lamin B1 forming the outer concentric ring located adjacent to the INM. The more peripheral localization of lamin B1 is mediated by its carboxyl-terminal farnesyl group. Lamin B1 localization is also curvature- and strain-dependent, while the localization of lamin A/C is not. We also show that lamin B1's outer-facing localization stabilizes nuclear shape by restraining outward protrusions of the lamin A/C network. These two findings, that lamin B1 forms an outer concentric ring and that its localization is energy-dependent, are significant as they suggest a distinct model for the nuclear lamina-one that is able to predict its behavior and clarifies the distinct roles of individual nuclear lamin proteins and the consequences of their perturbation.

Imbalanced nucleocytoskeletal connections create common polarity defects in progeria and physiological aging.

Studies of the accelerated aging disorder Hutchinson-Gilford progeria syndrome (HGPS) can potentially reveal cellular defects associated with physiological aging. HGPS results from expression and abnormal nuclear envelope association of a farnesylated, truncated variant of prelamin A called "progerin." We surveyed the diffusional mobilities of nuclear membrane proteins to identify proximal effects of progerin expression. The mobilities of three proteins-SUN2, nesprin-2G, and emerin-were reduced in fibroblasts from children with HGPS compared with those in normal fibroblasts. These proteins function together in nuclear movement and centrosome orientation in fibroblasts polarizing for migration. Both processes were impaired in fibroblasts from children with HGPS and in NIH 3T3 fibroblasts expressing progerin, but were restored by inhibiting protein farnesylation. Progerin affected both the coupling of the nucleus to actin cables and the oriented flow of the cables necessary for nuclear movement and centrosome orientation. Progerin overexpression increased levels of SUN1, which couples the nucleus to microtubules through nesprin-2G and dynein, and microtubule association with the nucleus. Reducing microtubule-nuclear connections through SUN1 depletion or dynein inhibition rescued the polarity defects. Nuclear movement and centrosome orientation were also defective in fibroblasts from normal individuals over 60 y, and both defects were rescued by reducing the increased level of SUN1 in these cells or inhibiting dynein. Our results identify imbalanced nuclear engagement of the cytoskeleton (microtubules: high; actin filaments: low) as the basis for intrinsic cell polarity defects in HGPS and physiological aging and suggest that rebalancing the connections can ameliorate the defects.

Beta-strand-mediated dimeric formation of the Ig-like domains of human lamin A/C and B1.

Lamins are nuclear intermediate filament proteins that play an essential role in maintaining the nuclear structure by forming a 3-D meshwork. Lamins consist of the N-terminal unstructured head, the coiled-coil rod domain, and the C-terminal tail, which is mostly unstructured except for the Ig-like domain. To date, the Ig-like domain has been characterized as a monomeric structure. Here, we determined the crystal structures of human lamin A/C, including the Ig-like domain and its N- and C-terminal flanking sequences. Interestingly, the structures showed a homodimer formed by beta-strand interactions between the N- and C-terminal flanking sequences. This interaction also provides a molecular implication for the creation of a 3-D meshwork between the 3.5-nm-thick filaments. Furthermore, we determined the crystal structure of the corresponding region of lamin B1. The structure showed a similar dimeric assembly, also formed by beta-strand interactions, albeit the intersubunit distance was much shorter. Since the Ig-like domain contains many genetic hotspots causing lamin-related diseases in lamin A/C, our findings will help understand the detailed assembly of lamins in a 3-D meshwork structure and lamin-related diseases at the molecular level.

HiPLA: High-throughput imaging proximity ligation assay.

Protein-protein interactions are essential for cellular structure and function. To delineate how the intricate assembly of protein interactions contribute to cellular processes in health and disease, new methodologies that are both highly sensitive and can be applied at large scale are needed. Here, we develop HiPLA (high-throughput imaging proximity ligation assay), a method that employs the well-established antibody-based proximity ligation assay in a high-throughput imaging screening format as a novel means to systematically visualize protein interactomes. Using HiPLA with a library of antibodies targeting nuclear proteins, we probe the interaction of 60 proteins and associated post-translational modifications (PTMs) with the nuclear lamina in a model of the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identify a subset of proteins that differentially interact with the nuclear lamina in HGPS. Using HiPLA in combination with quantitative indirect immunofluorescence, we find that the majority of differential interactions are accompanied by corresponding changes in expression of the interacting protein. Taken together, HiPLA offers a novel approach to probe cellular protein-protein interaction at a large scale and reveals mechanistic insights into the assembly of protein complexes.

Structural analysis of the ternary complex between lamin A/C, BAF and emerin identifies an interface disrupted in autosomal recessive progeroid diseases.

Lamins are the main components of the nucleoskeleton. Whereas their 3D organization was recently described using cryoelectron tomography, no structural data highlights how they interact with their partners at the interface between the inner nuclear envelope and chromatin. A large number of mutations causing rare genetic disorders called laminopathies were identified in the C-terminal globular Igfold domain of lamins A and C. We here present a first structural description of the interaction between the lamin A/C immunoglobulin-like domain and emerin, a nuclear envelope protein. We reveal that this lamin A/C domain both directly binds self-assembled emerin and interacts with monomeric emerin LEM domain through the dimeric chromatin-associated Barrier-to-Autointegration Factor (BAF) protein. Mutations causing autosomal recessive progeroid syndromes specifically impair proper binding of lamin A/C domain to BAF, thus destabilizing the link between lamin A/C and BAF in cells. Recent data revealed that, during nuclear assembly, BAF's ability to bridge distant DNA sites is essential for guiding membranes to form a single nucleus around the mitotic chromosome ensemble. Our results suggest that BAF interaction with lamin A/C also plays an essential role, and that mutations associated with progeroid syndromes leads to a dysregulation of BAF-mediated chromatin organization and gene expression.

A Nondimensional Model Reveals Alterations in Nuclear Mechanics upon Hepatitis C Virus Replication.

Morphology of the nucleus is an important regulator of gene expression. Nuclear morphology is in turn a function of the forces acting on it and the mechanical properties of the nuclear envelope. Here, we present a two-parameter, nondimensional mechanical model of the nucleus that reveals a relationship among nuclear shape parameters, such as projected area, surface area, and volume. Our model fits the morphology of individual nuclei and predicts the ratio between forces and modulus in each nucleus. We analyzed the changes in nuclear morphology of liver cells due to hepatitis C virus (HCV) infection using this model. The model predicted a decrease in the elastic modulus of the nuclear envelope and an increase in the pre-tension in cortical actin as the causes for the change in nuclear morphology. These predictions were validated biomechanically by showing that liver cells expressing HCV proteins possessed enhanced cellular stiffness and reduced nuclear stiffness. Concomitantly, cells expressing HCV proteins showed downregulation of lamin-A,C and upregulation of ß-actin, corroborating the predictions of the model. Our modeling assumptions are broadly applicable to adherent, monolayer cell cultures, making the model amenable to investigate changes in nuclear mechanics due to other stimuli by merely measuring nuclear morphology. Toward this, we present two techniques, graphical and numerical, to use our model for predicting physical changes in the nucleus.

Lamin A mutation impairs interaction with nucleoporin NUP155 and disrupts nucleocytoplasmic transport in atrial fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Here, we show the identification and functional characterization of one AF-associated mutation p.Arg399Cys in lamin A/C. Co-immunoprecipitation and GST pull-down assays demonstrate that lamin A/C interacts with NUP155, which is a nucleoporin and causes AF when mutated. Lamin A/C mutation p.Arg399Cys impairs the interaction between lamin A/C and NUP155, and increases extractability of NUP155 from the nuclear envelope (NE). Mutation p.Arg399Cys leads to aggregation of lamin A/C in the nucleus, although it does not impair the integrity of NE upon cellular stress. Mutation p.Arg399Cys inhibits the export of HSP70 mRNA and the nuclear import of HSP70 protein. Electrophysiological studies show that mutation p.Arg399Cys decreases the peak cardiac sodium current by decreasing the cell surface expression level of cardiac sodium channel Nav 1.5, but does not affect IKr potassium current. In conclusion, our results indicate that lamin A/C mutation p.Arg399Cys weakens the interaction between nuclear lamina (lamin A/C) and the nuclear pore complex (NUP155), leading to the development of AF. The findings provide a novel molecular mechanism for the pathogenesis of AF.

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.

Structural instability of lamin A tail domain modulates its assembly and higher order function in Emery-Dreifuss muscular dystrophy.

The C-terminal Ig-domain of lamin A plays critical roles in cell function via interaction with proteins, DNA, and chromatin. Mutations in this domain are known to cause various diseases including Emery-Dreifuss muscular dystrophy (EDMD) and familial partial lipodystrophy (FPLD). Here we examined the biophysical and biochemical properties of mutant Ig-domains identified in patients with EDMD and FPLD. EDMD-related mutant Ig-domain showed decreased stability to heat and denaturant. This result was also confirmed by experiments using full-length mutant lamin A, although the decrease in melting temperature was much less than that of the mutant Ig-domain alone. The unstable EDMD Ig-domain disrupted the proper assembly of lamin A, resulting in abnormal paracrystal formation and decreased viscosity. In contrast, FPLD-related mutant Ig-domains were thermally stable, although they lost DNA binding function. Alanine substitution experiments revealed a functional domain of DNA binding in the Ig-domain. Thus, the overall biophysical property of Ig-domains is closely associated with clinical phenotype.

Forced expression of mouse progerin attenuates the osteoblast differentiation interrupting ß-catenin signal pathway in vitro.

Nuclear protein, lamin A, which is a component of inner membrane on nucleoplasm, plays a role in nuclear formation and cell differentiation. The expression of mutated lamin A, termed progerin, causes a rare genetic aging disorder, Hutchinson-Gilford progeria syndrome, which shows abnormal bone formation with the decrease in a number of osteoblasts and osteocytes. However, exact molecular mechanism how progerin exerts depressive effects on osteogenesis has not been fully understood. Here, we created mouse lamin A dC50 cDNA encoding progerin that lacks 50 amino acid residues at C-terminus, transfected it in mouse preosteoblast-like MC3T3-E1 cells, and examined the changes in osteoblast phenotype. When lamin A dC50-expressed cells were cultured with differentiation-inductive medium, alkaline phosphatase (ALP) activity and mRNA levels of major osteoblast markers, type I collagen (Col1), bone sialoprotein (BSP), dentine matrix protein 1 (DMP1), and Runx2 were significantly decreased, and no mineralized nodules were detected as seen in control cells expressing empty vector. In the culture with mineralization-inductive medium, mRNA levels of BSP, osteocalcin, DMP1, Runx2, and osterix were strongly decreased parallel with loss of mineralization in lamin A dC50-expressed cells, while mineralized nodules appear at 21 days in control cells. Furthermore, lamin A dC50 expression was depressed nuclear localization of ß-catenin with the decrease of GSK-3ß phosphorylation level. These results suggest that lamin A dC50 depresses osteoblast differentiation in both early and late stages, and it negatively regulates ß-catenin activity interacting with GSK-3ß in cytoplasm.

The effect of the lamin A and its mutants on nuclear structure, cell proliferation, protein stability, and mobility in embryonic cells.

LMNA gene encodes for nuclear intermediate filament proteins lamin A/C. Mutations in this gene lead to a spectrum of genetic disorders, collectively referred to as laminopathies. Lamin A/C are widely expressed in most differentiated somatic cells but not in early embryos and some undifferentiated cells. To investigate the role of lamin A/C in cell phenotype maintenance and differentiation, which could be a determinant of the pathogenesis of laminopathies, we examined the role played by exogenous lamin A and its mutants in differentiated cell lines (HeLa, NHDF) and less-differentiated HEK 293 cells. We introduced exogenous wild-type and mutated (H222P, L263P, E358K D446V, and ∆50) lamin A into different cell types and analyzed proteins' impact on proliferation, protein mobility, and endogenous nuclear envelope protein distribution. The mutants give rise to a broad spectrum of nuclear phenotypes and relocate lamin C. The mutations ∆50 and D446V enhance proliferation in comparison to wild-type lamin A and control cells, but no changes in exogenous protein mobility measured by FRAP were observed. Interestingly, although transcripts for lamins A and C are at similar level in HEK 293 cells, only lamin C protein is detected in western blots. Also, exogenous lamin A and its mutants, when expressed in HEK 293 cells underwent posttranscriptional processing. Overall, our results provide new insight into the maintenance of lamin A in less-differentiated cells. Embryonic cells are very sensitive to lamin A imbalance, and its upregulation disturbs lamin C, which may influence gene expression and many regulatory pathways.

Emery-Dreifuss Muscular Dystrophy-Associated Mutant Forms of Lamin A Recruit the Stress Responsive Protein Ankrd2 into the Nucleus, Affecting the Cellular Response to Oxidative Stress.

BACKGROUND: Ankrd2 is a stress responsive protein mainly expressed in muscle cells. Upon the application of oxidative stress, Ankrd2 translocates into the nucleus where it regulates the activity of genes involved in cellular response to stress. Emery-Dreifuss Muscular Dystrophy 2 (EDMD2) is a muscular disorder caused by mutations of the gene encoding lamin A, LMNA. As well as many phenotypic abnormalities, EDMD2 muscle cells also feature a permanent basal stress state, the underlying molecular mechanisms of which are currently unclear. METHODS: Experiments were performed in EDMD2-lamin A overexpressing cell lines and EDMD2-affected human myotubes. Oxidative stress was produced by H2O2 treatment. Co-immunoprecipitation, cellular subfractionation and immunofluorescence analysis were used to validate the relation between Ankrd2 and forms of lamin A; cellular sensibility to stress was monitored by the analysis of Reactive Oxygen Species (ROS) release and cell viability. RESULTS: Our data demonstrate that oxidative stress induces the formation of a complex between Ankrd2 and lamin A. However, EDMD2-lamin A mutants were able to bind and mislocalize Ankrd2 in the nucleus even under basal conditions. Nonetheless, cells co-expressing Ankrd2 and EDMD2-lamin A mutants were more sensitive to oxidative stress than the Ankrd2-wild type lamin A counterpart. CONCLUSIONS: For the first time, we present evidence that in muscle fibers from patients affected by EDMD2, Ankrd2 has an unusual nuclear localization. By introducing a plausible mechanism ruling this accumulation, our data hint at a novel function of Ankrd2 in the pathogenesis of EDMD2-affected cells.

DCM associated LMNA mutations cause distortions in lamina structure and assembly.

BACKGROUND: A and B-type lamins are integral scaffolding components of the nuclear lamina which impart rigidity and shape to all metazoan nuclei. Over 450 mutations in A-type lamins are associated with 16 human diseases including dilated cardiomyopathy (DCM). Here, we show that DCM mutants perturb the self-association of lamin A (LA) and it's binding with lamin B1 (LB1). METHODS: We used confocal and superresolution microscopy (NSIM) to study the effect of LA mutants on the nuclear lamina. We further used circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry (ITC) to probe the structural modulations, self-association and heteropolymeric association of mutant LA. RESULTS: Transfection of mutants in cultured cell lines result in the formation of nuclear aggregates of varied size and distribution. Endogenous LB1 is sequestered into these aggregates. This is consistent with the ten-fold increase in association constant of the mutant proteins compared to the wild type. These mutants exhibit differential heterotypic interaction with LB1, along with significant secondary and tertiary structural alterations of the interacting proteins. Thermodynamic studies demonstrate that the mutants bind to LB1 with different stoichiometry, affinity and energetics. CONCLUSIONS: In this report we show that increased self-association propensity of mutant LA modulates the LA-LB1 interaction and precludes the formation of an otherwise uniform laminar network. GENERAL SIGNIFICANCE: Our results might highlight the role of homotypic and heterotypic interactions of LA in the pathogenesis of DCM and hence laminopathies in the broader sense.

Lamin A reassembly at the end of mitosis is regulated by its SUMO-interacting motif.

Modification of proteins with small ubiquitin-related modifier (SUMO; SUMOylation) is involved in the regulation of various biological processes. Recent studies have demonstrated that noncovalent associations between SUMOylated proteins and co-operative proteins containing SUMO-interacting motifs (SIMs) are important for the spatiotemporal organization of many protein complexes. In this study, we demonstrate that interactions between lamin A, a major component of the nuclear lamina, and SUMO isoforms are dependent on one of the four SIMs (SIM3) resided in lamin A polypeptide in vitro. Live cell imaging and immunofluorescence staining showed that SIM3 is required for accumulation of lamin A on the chromosomes during telophase, and subsequent evaluation of a panel of deletion mutants determined that a 156-amino acid region spanning the carboxyl-terminal Ig-fold domain of lamin A is sufficient for this accumulation. Notably, mutation of SIM3 abrogated the dephosphorylation of mitosis-specific phosphorylation at Ser-22 of lamin A, which normally occurs during telophase, and the subsequent nuclear lamina reorganization. Furthermore, expression of a conjugation-defective SUMO2 mutant, which was previously shown to inhibit endogenous SUMOylation in a dominant-negative manner, also impaired the accumulation of wild type lamin A on telophase chromosomes. These findings suggest that interactions between SIM3 of lamin A and a putative SUMO2-modified protein plays an important role in the reorganization of the nuclear lamina at the end of mitosis.

Deregulation of Fragile X-related protein 1 by the lipodystrophic lamin A p.R482W mutation elicits a myogenic gene expression program in preadipocytes.

The nuclear lamina is implicated in the regulation of various nuclear functions. Several laminopathy-causing mutations in the LMNA gene, notably the p.R482W substitution linked to familial partial lipodystrophy type 2 (FPLD2), are clustered in the immunoglobulin fold of lamin A. We report a functional association between lamin A and fragile X-related protein 1 (FXR1P), a protein of the fragile X-related family involved in fragile X syndrome. Searching for proteins differentially interacting with the immunoglobulin fold of wild-type and R482W mutant lamin A, we identify FXR1P as a novel component of the lamin A protein network. The p.R482W mutation abrogates interaction of FXR1P with lamin A. Fibroblasts from FPLD2 patients display elevated levels of FXR1P and delocalized FXR1P. In human adipocyte progenitors, deregulation of lamin A expression leads to FXR1P up-regulation, impairment of adipogenic differentiation and induction of myogenin expression. FXR1P overexpression also stimulates a myogenic gene expression program in these cells. Our results demonstrate a cross-talk between proteins hitherto implicated in two distinct mesodermal pathologies. We propose a model where the FPLD2 lamin A p.R482W mutation elicits, through up-regulation of FXR1P, a remodeling of an adipogenic differentiation program into a myogenic program.