Bertrand Favre

Analysis of the interactions between BP180, BP230, plectin and the integrin alpha6beta4 important for hemidesmosome assembly.

Hemidesmosomes (HDs) are multi-protein complexes that promote stable adhesion of epithelial cells to the underlying extracellular matrix. We assessed the interactions between different hemidesmosomal components with each other, mapped the binding sites and studied the importance of these interactions for HD assembly in yeast two-hybrid and cell-transfection assays. The results show that: (1) bullous pemphigoid antigen (BP) 180 binds not only to BP230, but also to plectin. The interactions between these proteins are facilitated by the Y subdomain in the N-terminal plakin domain of BP230 and plectin, and residues 145-230 of the cytoplasmic domain of BP180; (2) different, but overlapping, sequences on BP180 mediate binding to β4, which, in turn associates with BP180 via its third fibronectin type III repeat; (3) sequences in the N-terminal extremity of BP230 mediate its binding to β4, which requires the C-terminal end of the connecting segment up to the fourth FNIII repeat of the β4 subunit. (4) Finally, cell-transfection studies showed that the localization of BP230 into hemidesmosome-like structures depends on its Z-Y subdomains as well as on the availability of BP180. By having further uncovered interactions between various hemidesmosomal components, mapped the involved binding sites and dissected a hierarchy of interactions relevant for their topogenic fate, our findings give novel insights into the molecular organization of hemidesmosomes.

The Protease Inhibitor Alpha-2-Macroglobuline-Like-1 Is the p170 Antigen Recognized by Paraneoplastic Pemphigus Autoantibodies in Human

Background Paraneoplastic pemphigus (PNP) is a devastating autoimmune blistering disease, involving mucocutaneous and internal organs, and associated with underlying neoplasms. PNP is characterized by the production of autoantibodies targeting proteins of the plakin and cadherin families involved in maintenance of cell architecture and tissue cohesion. Nevertheless, the identity of an antigen of Mr 170,000 (p170), thought to be critical in PNP pathogenesis, has remained unknown. Methodology/Principal Findings Using an immunoprecipitation and mass spectrometry based approach, we identified p170 as alpha-2-macroglobuline-like-1, a broad range protease inhibitor expressed in stratified epithelia and other tissues damaged in the PNP disease course. We demonstrate that 10 PNP sera recognize alpha-2-macroglobuline-like-1 (A2ML1), while none of the control sera obtained from patients with bullous pemphigoid, pemphigus vulgaris, pemphigus foliaceus and normal subjects does. Conclusions/Significance Our study unravels a broad range protease inhibitor as a new class of target antigens in a paraneoplastic autoimmune multiorgan syndrome and opens a new challenging investigation avenue for a better understanding of PNP pathogenesis.

Plakins, a Versatile Family of Cytolinkers: Roles in Skin Integrity and in Human Diseases

The plakin family consists of giant proteins involved in the cross-linking and organization of the cytoskeleton and adhesion complexes. They further modulate several fundamental biological processes, such as cell adhesion, migration, and polarization or signaling pathways. Inherited and acquired defects of plakins in humans and in animal models potentially lead to dramatic manifestations in the skin, striated muscles, and/or nervous system. These observations unequivocally demonstrate the key role of plakins in the maintenance of tissue integrity. Here we review the characteristics of the mammalian plakin members BPAG1 (bullous pemphigoid antigen 1), desmoplakin, plectin, envoplakin, epiplakin, MACF1 (microtubule-actin cross-linking factor 1), and periplakin, highlighting their role in skin homeostasis and diseases.

New insights into the molecular basis of desmoplakin- and desmin-related cardiomyopathies

Desmosomes are intercellular adhesive complexes that anchor the intermediate filament cytoskeleton to the cell membrane in epithelia and cardiac muscle cells. The desmosomal component desmoplakin plays a key role in tethering various intermediate filament networks through its C-terminal plakin repeat domain. To gain better insight into the cytoskeletal organization of cardiomyocytes, we investigated the association of desmoplakin with desmin by cell transfection, yeast two-hybrid, and/or in vitro binding assays. The results indicate that the association of desmoplakin with desmin depends on sequences within the linker region and C-terminal extremity of desmoplakin, where the B and C subdomains contribute to efficient binding; a potentially phosphorylatable serine residue in the C-terminal extremity of desmoplakin affects its association with desmin; the interaction of desmoplakin with non-filamentous desmin requires sequences contained within the desmin C-terminal rod portion and tail domain in yeast, whereas in in vitro binding studies the desmin tail is dispensable for association; and mutations in either the C-terminus of desmoplakin or the desmin tail linked to inherited cardiomyopathy seem to impair desmoplakindesmin interaction. These studies increase our understanding of desmoplakin-intermediate filament interactions, which are important for maintenance of cytoarchitecture in cardiomyocytes, and give new insights into the molecular basis of desmoplakin- and desmin-related human diseases.

Identification of a calcium- and phospholipid-dependent protein kinase (protein kinase C) in Neurospora crassa

Abstract Protein kinase C was partially purified by DEAE-cellulose chromatography from the soluble fraction of mycelium of Neurospora crassa. In the presence of calcium phosphatidylserine was found to be the best activator among the various lipids tested. Diacylglycerol, under our experimental conditions, affected neither the K m of the enzyme for calcium nor the basal or maximum enzymatic activity. Two endogenous substrates were copurified with the enzyme and were better phosphate acceptor than other exogenous basic or acidic proteins tested. The largest phosphorylated band observed on sodium dodecylsulfate-polyacrylamide gel had an apparent molecular weight of 85 000 daltons. The M r of the other was smaller than 14 000.

Autoantibodies to the extracellular and intracellular domain of bullous pemphigoid 180, the putative key autoantigen in bullous pemphigoid, belong predominantly to the IgG1 and IgG4 subclasses

Background Autoantibodies to the extracellular domain (ECD) of bullous pemphigoid (BP) antigen 180 (BP180) are thought to play a crucial part in the pathophysiology of BP.

Plectin interacts with the rod domain of type III intermediate filament proteins desmin and vimentin

Plectin is a versatile cytolinker protein critically involved in the organization of the cytoskeletal filamentous system. The muscle-specific intermediate filament (IF) protein desmin, which progressively replaces vimentin during differentiation of myoblasts, is one of the important binding partners of plectin in mature muscle. Defects of either plectin or desmin cause muscular dystrophies. By cell transfection studies, yeast two-hybrid, overlay and pull-down assays for binding analysis, we have characterized the functionally important sequences for the interaction of plectin with desmin and vimentin. The association of plectin with both desmin and vimentin predominantly depended on its fifth plakin repeat domain and downstream linker region. Conversely, the interaction of desmin and vimentin with plectin required sequences contained within the segments 1A-2A of their central coiled-coil rod domain. This study furthers our knowledge of the interaction between plectin and IF proteins important for maintenance of cytoarchitecture in skeletal muscle. Moreover, binding of plectin to the conserved rod domain of IF proteins could well explain its broad interaction with most types of IFs.

Plectin, an unusual target antigen in bullous pemphigoid

Background  Bullous pemphigoid (BP) is a blistering disease associated with autoantibodies directed against two components of hemidesmosomes, BP180 and BP230.

One gene but different proteins and diseases: the complexity of dystonin and bullous pemphigoid antigen 1

Since the immunochemical identification of the bullous pemphigoid antigen 230 (BP230) as one of the major target autoantigens of bullous pemphigoid (BP) in 1981, our understanding of this protein has significantly increased. Cloning of its gene, development and characterization of animal models with engineered gene mutations or spontaneous mouse mutations have revealed an unexpected complexity of the gene encoding BP230. The latter, now called dystonin (DST), is composed of at least 100 exons and gives rise to three major isoforms, an epithelial, a neuronal and a muscular isoform, named BPAG1e (corresponding to the original BP230), BPAG1a and BPAG1b, respectively. The various BPAG1 isoforms play a key role in fundamental processes, such as cell adhesion, cytoskeleton organization, and cell migration. Genetic defects of BPAG1 isoforms are the culprits of epidermolysis bullosa and complex, devastating neurological diseases. In this review, we summarize recent advances of our knowledge about several BPAG1 isoforms, their role in various biological processes and in human diseases.

BPAG1a and b Associate with EB1 and EB3 and Modulate Vesicular Transport, Golgi Apparatus Structure, and Cell Migration in C2.7 Myoblasts

BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5′ end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3′ end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts.