Structural and functional analysis of lamina-associated polypeptide 2 alpha complexes and its implication for human disease

Lamina-associated polypeptide 2 alpha (LAP2a) is the only protein of the LAP2 family, which has been found in nucleoskeletal structures throughout the nuclear interior. It is likely involved in the dynamics of higher order chromatin organizatioin during cell proliferation and cell differentiation. LAP2a dynamically associates with chromosomes during cell division and interacts specifically with A-type lamins. Recent observations that mutations in the lamin A gene cause various inheritable human diseases (laminopathies) involving muscle and fat tissue, have underlined the fundamental functions of lamin A and lamin A-interacting proteins such as LAP2a, but the molecular mechanisms remain unknown. According to one model, mutations in lamin A may cause defects in structural lamin complexes making the nuclear scaffold more susceptible to mechanical stress. In a second model, lamin filaments and proteins such as LAP2a may provide assembly sites for specific transcriptional regulators. Our proposed work will address fundamental questions about the cellular functions of the lamin A - LAP2a complex and attempts to unravel the potential involvement of LAP2a in human disease. To analyze the structural role of LAP2a we will investigate the molecular structure of LAP2a -homo-oligomeres and LAP2a - lamin A complexes by rotary shadowing and negative staining electron microscopy. Furthermore, we will investigate how disease-specific mutations in lamin A effect lamin A - LAP2a complexes on the structural and functional level in vitro and in vivo upon expression of mutated proteins. We will also elucidate the role of cysteine residues in determining LAP2a structure by mutating cysteins to alanine or serine. Yeast-two-hybrid analysis using HeLa and muscle specific libraries, as well as affinity chromatography of muscle specific proteins on immobilized LAP2a and lamin A, combined with MALDI-TOF mass spectrometry will identify novel general or muscle specific interaction partners of LAP2a and lamin A. These will be characterized in detail using cell biological and biochemical approaches. We will also perform conditional LAP2a knockouts in various tissues in mice and test mice for laminopathy-related phenotypes. The results of this study should provide novel insights in disease related mechanisms and set the basis for the development of diagnostic and therapeutic tools and strategies.

Lamins are important proteins in the nucleus of mammalian cells and are involved in many essential processes including gene replication and control of gene expression during tissue development. Some types of lamins, termed B-type lamins are essential for early embryonic development, others, the A-type lamins are important in the adult organism. The latter proteins have been linked to rare diseases, which are clinically manifested only after birth during childhood and can affect heart, muscle, fat, bone, and neuronal tissues or cause accelerated ageing syndromes. Despite the importance of lamins in development and diseases, very little is known about their molecular functions and how these functions are altered in patients suffering from these diseases. Yet this knowledge is essential for the development of improved diagnostic and therapeutic approaches. One approach to obtain new information on the functions of lamins is to identify and analyze specific lamin binding partners. We have been focusing on a protein, Lamina-associated polypeptide 2alpha (LAP2a), which specifically interacts with A-type lamins and may be involved in disease mechanisms. Our studies revealed that LAP2a has at least two different functions in mammalian cells. One function, which might be independent of A-type lamins, is the dynamic organisation of chromatin during cell division, an important process ensuring the equal distribution of genetic information between daughter cells. We discovered another unexpected role of the protein in controlling the balance between cell proliferation and differentiation. This process is involved in the regulation of the turnover, repair and regeneration of many tissues from adult stem cells. Our studies in mammalian cell cultures indicated that complexes of A-type lamins and LAP2a are required for an efficient differentiation of adult stem cells into tissue- specific cells. We also generated a transgenic mouse model, in which the gene encoding LAP2a was deleted. Initial analysis of LAP2a-deficient mice indicated that stressed tissues with a high degree of turnover show an abnormal phenotype, supporting our hypothesis. Our findings have major implications for understanding the molecular basis of lamin-linked diseases. In contrast to previously suggested disease hypotheses, assuming a degeneration of affected tissues in patients, our studies indicate a less effective tissue regeneration through a defect in adult stem cell differentiation.