Lamin A/C, laminopathies and premature ageing

Lamin A/C belongs to type V intermediate filaments and constitutes the nuclear lamina and nuclear matrix, where a variety of nuclear activities occur. Lamin A/C protein is firstly synthesized as a precursor and is further proteolytically processed by the zinc metallo-proteinase Ste24 (Zmpste24). Lamin A/C mutations cause a series of human diseases, collectively called laminopathies, the most severe of which is Hutchinson Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD) which arises due to an unsuccessful maturation of prelamin A. Although the exact underlying molecular mechanisms are still poorly understood, genomic instability, defective nuclear mechanics and mechanotransduction, have been hypothesized to be responsible for laminopathy-based premature ageing. Removal of unprocessed prelamin A (progerin) or rescue of defective DNA repair could be potential therapeutic strategies for the treatment of HGPS in future.     

A nuclear lamina‐chromatin‐Ran GTPase axis modulates nuclear import and DNA damage signaling

The Ran GTPase regulates nuclear import and export by controlling the assembly state of transport complexes. This involves the direct action of RanGTP, which is generated in the nucleus by the chromatin‐associated nucleotide exchange factor, RCC1. Ran interactions with RCC1 contribute to formation of a nuclear:cytoplasmic (N:C) Ran protein gradient in interphase cells. In previous work, we showed that the Ran protein gradient is disrupted in fibroblasts from Hutchinson–Gilford progeria syndrome (HGPS) patients. The Ran gradient disruption in these cells is caused by nuclear membrane association of a mutant form of Lamin A, which induces a global reduction in heterochromatin marked with Histone H3K9me3 and Histone H3K27me3. Here, we have tested the hypothesis that heterochromatin controls the Ran gradient. Chemical inhibition and depletion of the histone methyltransferases (HMTs) G9a and GLP in normal human fibroblasts reduced heterochromatin levels and caused disruption of the Ran gradient, comparable to that observed previously in HGPS fibroblasts. HMT inhibition caused a defect in nuclear localization of TPR, a high molecular weight protein that, owing to its large size, displays a Ran‐dependent import defect in HGPS. We reasoned that pathways dependent on nuclear import of large proteins might be compromised in HGPS. We found that nuclear import of ATM requires the Ran gradient, and disruption of the Ran gradient in HGPS causes a defect in generating nuclear γ‐H2AX in response to ionizing radiation. Our data suggest a lamina–chromatin–Ran axis is important for nuclear transport regulation and contributes to the DNA damage response.     

Association of lamin A/C with muscle gene-specific promoters in myoblasts

The A-type and B-type lamins form a filamentous meshwork underneath the inner nuclear membrane called the nuclear lamina, which is an important component of nuclear architecture in metazoan cells. The lamina interacts with large, mostly repressive chromatin domains at the nuclear periphery. In addition, genome–lamina interactions also involve dynamic association of lamin A/C with gene promoters in adipocytes. Mutations in the human lamin A gene cause a spectrum of hereditary diseases called the laminopathies which affect muscle, cardiac and adipose tissues. Since most mutations in lamin A/C affect skeletal muscle, we investigated lamin–chromatin interactions at promoters of muscle specific genes in both muscle and non-muscle cell lines by ChIP-qPCR. We observed that lamin A/C was specifically associated with promoter regions of muscle genes in myoblasts but not in fibroblasts. Lamin A/C dissociated from the promoter regions of the differentiation specific MyoD, myogenin and muscle creatine kinase genes when myoblasts were induced to differentiate. In the promoter regions of the myogenin and MyoD genes, the binding of lamin A/C in myoblasts inversely correlated with the active histone mark, H3K4me3. Lamin A/C binding on muscle genes was reduced and differentiation potential was enhanced on treatment of myoblasts with a histone deacetylase inhibitor. These findings suggest a role for lamina–chromatin interactions in muscle differentiation and have important implications for the pathological mechanisms of striated muscle associated laminopathies.     

Correction of cellular phenotypes of Hutchinson-Gilford Progeria cells by RNA interference

The great majority of cases of the Hutchinson-Gilford progeroid syndrome (HGPS) (“Progeria of Childhood‘’) are caused by a single nucleotide mutation (1824 C->T) in the LMNA gene which encodes lamin A and C, nuclear intermediate filaments that are important components of the nuclear lamina. The resultant mutant protein (Δ50 lamin A) is thought to act in a dominant fashion. We exploited RNA interference technology to suppress Δ50 lamin A expression, with the long range goal of intervening in the pathogenesis of the coronary artery atherosclerosis that typically leads to the death of HGPS patients. Short hairpin RNA (shRNA) constructs were designed to target the mutated pre-spliced or mature LMNA mRNAs, and were expressed in HGPS fibroblasts carrying the 1824 C->T mutations using lentiviruses. One of the shRNAs targeted to the mutated mRNA reduced the expression levels of Δ50 lamin A to 26% or lower. The reduced expression was associated with amelioration of abnormal nuclear morphology, improvement of proliferative potential, and reduction in the numbers of senescent cells. These findings provide a rationale for potential gene therapy.     

Genomic instability in laminopathy-based premature aging

Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24-/- mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24-/- MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.     

From lamins to lamina: a structural perspective

Lamin proteins are the major constituents of the nuclear lamina, a proteinaceous network that lines the inner nuclear membrane. Primarily, the nuclear lamina provides structural support for the nucleus and the nuclear envelope; however, lamins and their associated proteins are also involved in most of the nuclear processes, including DNA replication and repair, regulation of gene expression, and signaling. Mutations in human lamin A and associated proteins were found to cause a large number of diseases, termed ‘laminopathies.’ These diseases include muscular dystrophies, lipodystrophies, neuropathies, and premature aging syndromes. Despite the growing number of studies on lamins and their associated proteins, the molecular organization of lamins in health and disease is still elusive. Likewise, there is no comprehensive view how mutations in lamins result in a plethora of diseases, selectively affecting different tissues. Here, we discuss some of the structural aspects of lamins and the nuclear lamina organization, in light of recent results.     

Mouse models of the laminopathies

The A and B type lamins are nuclear intermediate filament proteins that comprise the bulk of the nuclear lamina, a thin proteinaceous structure underlying the inner nuclear membrane. The A type lamins are encoded by the lamin A gene (LMNA). Mutations in this gene have been linked to at least nine diseases, including the progeroid diseases Hutchinson-Gilford progeria and atypical Werner's syndromes, striated muscle diseases including muscular dystrophies and dilated cardiomyopathies, lipodystrophies affecting adipose tissue deposition, diseases affecting skeletal development, and a peripheral neuropathy. To understand how different diseases arise from different mutations in the same gene, mouse lines carrying some of the same mutations found in the human diseases have been established. We, and others have generated mice with different mutations that result in progeria, muscular dystrophy, and dilated cardiomyopathy. To further our understanding of the functions of the lamins, we also created mice lacking lamin B1, as well as mice expressing only one of the A type lamins. These mouse lines are providing insights into the functions of the lamina and how changes to the lamina affect the mechanical integrity of the nucleus as well as signaling pathways that, when disrupted, may contribute to the disease.     

Folding at the rhythm of the rare codon beat

The persistent difficulties in the production of protein at high levels in heterologous systems, as well as the inability to understand pathologies associated with protein aggregation, highlight our limited knowledge on the mechanisms of protein folding in vivo. Attempts to improve yield and quality of recombinant proteins are diverse, frequently involving optimization of the cell growth temperature, the use of synonymous codons and/or the co-expression of tRNAs, chaperones and folding catalysts among others. Although protein secondary structure can be determined largely by the amino acid sequence, protein folding within the cell is affected by a range of factors beyond amino acid sequence. The folding pathway of a nascent polypeptide can be affected by transient interactions with other proteins and ligands, the ribosome, translocation through a pore membrane, redox conditions, among others. The translation rate as well as the translation machinery itself can dramatically affect protein folding, and thus the structure and function of the protein product. This review addresses current efforts to better understand how the use of synonymous codons in the mRNA and the availability of tRNAs can modulate translation kinetics, affecting the folding, the structure and the biological activity of proteins.     

A-type lamins and progeroïd syndromes : persistent farnesylation with dramatic effects

Hutchinson-Gilford Progeria (HGPS), a rare and severe developmental disorder characterized by features recalling premature aging, and Restrictive Dermopathy (RD), a neonatal lethal genodermatosis, have recently been identified as being primary or secondary < Laminopathies >. These heterogeneous disorders are caused by altered Lamin maturation pathway. In physiological conditions, mature Lamin A is obtained through a series of post-translational processing steps performed on a protein precursor, Prelamin A. The major pathophysiological mechanism involved in Progeria is an aberrant splicing due to a de novo heterozygous point mutation, leading to the accumulation of truncated Lamin A precursor. The same aberrant splicing mechanism was involved in RD, whereas the majority of RD cases are caused by ZMPSTE24/FACE1 inactivation, a key enzyme involved in the Lamin A maturation pathway. In functional terms, all these conditions share the same pathophysiological mechanism, i.e. the intranuclear accumulation of Lamin A precursors, which cannot be fully processed and exert a toxic effect on nuclear homeostasis. In this article, we review the structure and functions of A-type Lamins, focusing namely on HGPS, RD or MAD disorders, in relation to existing animal models and possible future therapeutic approaches.     

Identification of cyclin D3 as a new interaction partner of lamin A/C

Lamin A/C is a major component of the nuclear lamina. An intact nuclear lamina has been proposed to be necessary for muscle differentiation. Cyclin D3 is known to be upregulated in differentiated muscle cells and to form insoluble complexes with cell-cycle regulatory factors in these cells. We have examined the possibility of direct binding interactions between lamin A/C and cyclin D3 by in vitro binding assays and co-immunoprecipitation studies with muscle cells. Our results indicate that cyclin D3 binds specifically to amino acid residues 383-474 of lamin A/C and associates with lamin A/C in muscle cells. The identification of cyclin D3 as a novel binding partner of lamin A/C has important implications for a role for lamin A/C in muscle differentiation.     

Structure modeling and antidiabetic activity of a seed protein of Momordica charantia in non-obese diabetic (NOD) mice

Momordica charantia is a well known medicinal plant used in the traditional medicinal system for the treatment of various diseases including diabetes mellitus. Recently, a novel protein termed as ADMc1 from the seed extract of M. charantia has been identified and isolated showing significant antihyperglycemic activity in type 1 diabetic rats in which diabetes was induced. However, the structure of this protein has not yet been analyzed. Homology modeling approach was used to generate a high quality protein 3D structure for the amino acid sequence of the ADMc1 protein in this study. The comparative assessment of secondary structures revealed ADMc1 as an all-alpha helix protein with random coils. Tertiary structure predicted on the template structure of Napin of B. Napus (PDB ID: 1SM7) with which the ADMc1 showed significant sequence similarity, was validated using protein structure validation tools like PROCHECK, WHAT_CHECK, VERIFY3D and ProSA. Arrangement of disulfide bridges formed by cysteine residues were predicted by the Dianna 1.1 server. The presence of multiple disulfide bond confers the stable nature of the ADMc1 protein. Further, the biological activity of the ADMc1 was assessed in non-obese diabetic (NOD) mice which are spontaneous model of type 1 diabetes. Significant reduction in the blood glucose levels of NOD mice was observed up to 8 h post administration of the rADMc1 protein. Overall, the structural characterizations with antihyperglycemic activity of this seed protein of Momordica charantia demonstrate its potential as an antidiabetic agent.     

Muscle dystrophy single point mutation in the 2B segment of lamin A does not affect the mechanical properties at the dimer level

Lamin intermediate filaments at the inner nuclear membrane play a key role in mechanosensation and gene regulation processes, and further guarantee the mechanical stability of the cell's nucleus. The rod-like dimers are the elementary building blocks within the dense lamina meshwork, mainly consisting of four alpha-helical coiled-coil segments as fundamental building blocks. Several mutations in the 2B segment of the rod domain of lamin A have been linked to the disease muscle dystrophy. In these diseases, the cell nuclei have been shown to feature abnormalities in the shape and its mechanical properties, leading to torn nuclear envelopes or bleb formation. However, up to now the origin of these mechanical changes remains unknown, in particular whether or not the mutations in the rod domain influence the mechanical properties of individual dimers, or if the changes are due to effects at larger hierarchical scales. Here we report a series of large-scale molecular dynamics studies of lamin A dimer segments, systematically comparing the mechanical behavior of the wild-type protein structure and a missense mutated protein structure with the point mutation p.Glu358Lys. Our results show that the nanomechanical tensile behavior of the dimer segment does not vary under presence of this mutation, suggesting that this single point mutation in muscle dystrophy does not affect the mechanical properties of lamin at the dimer level, but probably influences higher hierarchical scales.     

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

Dilated cardiomyopathy (DCM) is a condition whereby the normal muscular function of the myocardium is altered by specific or multiple aetiologies. About 25-35% of DCM patients show familial forms of the disease, with most mutations affecting genes encoding cytoskeletal proteins. Most of the DCM-related mutations fall in the Lamin AC gene, in particular in the Coil2B domain of the encoded protein. In this context, we focussed our studies on the crystal structures of two lamin Coil2B domain mutants (R335W and E347K). Both R335 and E347 are higly conserved residues whose substitution has little effects on the Coil2B domain three-dimensional structure; we can thus hypothesize that the mutations may interfere with the binding of components within the nuclear lamina, or of nuclear factors, that have been proposed to interact/associate with lamin A/C.     

Influence of Lamin A on the Mechanical Properties of Amphibian Oocyte Nuclei Measured by Atomic Force Microscopy

The nuclear lamina is part of the nuclear envelope (NE). Lamin filaments provide the nucleus with mechanical stability and are involved in many nuclear activities. The functional importance of these proteins is highlighted by mutations in lamin genes, which cause a variety of human diseases (laminopathies). Here we describe a method that allows one to quantify the contribution of lamin A protein to the mechanical properties of the NE. Lamin A is ectopically expressed in Xenopus oocytes, where it is incorporated into the NE of the oocyte nucleus, giving rise to a prominent lamina layer at the inner nuclear membrane. Nuclei are then isolated and probed by atomic force microscopy. From the resulting force curves, stiffness values are calculated and compared with those of control nuclei. Expression of lamin A significantly increases the stiffness of oocyte nuclei in a concentration-dependent manner. Since chromatin adds negligibly to nuclear mechanics in these giant nuclei, this method allows one to measure the contribution of individual NE components to nuclear mechanics.     

Protein design from in silico dynamic information: the emergence of the 'turn-dock-lock' motif

A protein design methodology based on ab initio folding simulations is described and illustrated. First, the time evolution of the chain topology is generated to identify a collapse-triggering nucleus. Then, a minimal spliced sequence of nuclear residues is created and systematically mutated in silico until it can sustain a stable conformation retaining the original nucleus topology. The mutations introduce a structural compensation for the deletions and eventually lead to the recovery of the native fold motif beyond topological identity. For ubiquitin, the systematically modified sequence is predicted to be a resilient folder, since it is 92% homologous to the hyperthermophile variant of B1-domain in streptococcal protein G. The methodology enabling us to identify the nucleus is independently validated vis-á-vis site-directed mutagenesis experiments on chymotrypsin inhibitor (CI2).     

Mammalian SUN Protein Interaction Networks at the Inner Nuclear Membrane and Their Role in Laminopathy Disease Processes

The nuclear envelope (NE) LINC complex, in mammals comprised of SUN domain and nesprin proteins, provides a direct connection between the nuclear lamina and the cytoskeleton, which contributes to nuclear positioning and cellular rigidity. SUN1 and SUN2 interact with lamin A, but lamin A is only required for NE localization of SUN2, and it remains unclear how SUN1 is anchored. Here, we identify emerin and short nesprin-2 isoforms as novel nucleoplasmic binding partners of SUN1/2. These have overlapping binding sites distinct from the lamin A binding site. However, we demonstrate that tight association of SUN1 with the nuclear lamina depends upon a short motif within residues 209–228, a region that does not interact significantly with known SUN1 binding partners. Moreover, SUN1 localizes correctly in cells lacking emerin. Importantly then, the major determinant of SUN1 NE localization has yet to be identified. We further find that a subset of lamin A mutations, associated with laminopathies Emery-Dreifuss muscular dystrophy (EDMD) and Hutchinson-Gilford progeria syndrome (HGPS), disrupt lamin A interaction with SUN1 and SUN2. Despite this, NE localization of SUN1 and SUN2 is not impaired in cell lines from either class of patients. Intriguingly, SUN1 expression at the NE is instead enhanced in a significant proportion of HGPS but not EDMD cells and strongly correlates with pre-lamin A accumulation due to preferential interaction of SUN1 with pre-lamin A. We propose that these different perturbations in lamin A-SUN protein interactions may underlie the opposing effects of EDMD and HGPS mutations on nuclear and cellular mechanics.     

Nuclear envelope transmembrane proteins (NETs) that are up-regulated during myogenesis

Background  

The nuclear lamina is a protein meshwork lining the inner nuclear membrane, which contains a polymer of nuclear lamins associated with transmembrane proteins of the inner nuclear membrane. The lamina is involved in nuclear structure, gene expression, and association of the cytoplasmic cytoskeleton with the nucleus. We previously identified a group of 67 novel putative nuclear envelope transmembrane proteins (NETs) in a large-scale proteomics analysis. Because mutations in lamina proteins have been linked to several human diseases affecting skeletal muscle, we examined NET expression during differentiation of C2C12 myoblasts. Our goal was to identify new nuclear envelope and lamina components whose expression is coordinated with muscle differentiation.     

A-type lamin complexes and regenerative potential: a step towards understanding laminopathic diseases?

The lamins are nuclear intermediate filament-type proteins forming the nuclear lamina meshwork at the inner nuclear membrane as well as complexes in the nucleoplasm. The recent discoveries that mutated A-type lamins and lamin-binding nuclear membrane proteins can be linked to numerous rare human diseases (laminopathies) affecting a multitude of tissues has changed the cell biologist’s view of lamins as mere structural nuclear scaffold proteins. It is still unclear how mutations in these ubiquitously expressed proteins give rise to tissue-restricted pathological phenotypes. Potential disease models include mutation-caused defects in lamin structure and stability, the deregulation of gene expression, and impaired cell cycle control. This review brings together various previously proposed ideas and suggests a novel, more general, disease model based on an impairment of adult stem cell function and thus compromised tissue regeneration in laminopathic diseases.