In-depth analysis of hyaline fibromatosis syndrome frameshift mutations at the same site reveal the necessity of personalized therapy

Hyaline fibromatosis syndrome is an autosomal recessive disease caused by mutations in ANTXR2, a gene involved in extracellular matrix homeostasis. Sixty percent of patients carry frameshift mutations at a mutational hotspot in exon 13. We show in patient cells that these mutations lead to low ANTXR2 mRNA and undetectable protein levels. Ectopic expression of the proteins encoded by the mutated genes reveals that a two base insertion leads to the synthesis of a protein that is rapidly targeted to the ER-associated degradation pathway due to the modified structure of the cytosolic tail, which instead of being hydrophilic and highly disordered as in wild type ANTXR2, is folded and exposes hydrophobic patches. In contrast, one base insertion leads to a truncated protein that properly localizes to the plasma membrane and retains partial function. We next show that targeting the nonsense mediated mRNA decay pathway in patient cells leads to a rescue of ANTXR2 protein in patients carrying one base insertion but not in those carrying two base insertions. This study highlights the importance of in-depth analysis of the molecular consequences of specific patient mutations, which even when they occur at the same site can have drastically different consequences.

From polymers to gene regulation

Aleksandre Japaridze

DNA molecule is the fundamental component of every living organism, since it encodes the hereditary information. Although the genetic code of almost 200 species has been sequenced, the direct connection between gene sequence, DNA structure and biological function remains poorly understood. The aim of this thesis was to investigate the connection between DNA function and structure. Throughout the thesis we will discuss experiments testing the link between different physical properties of DNA with its biological function. For this purpose we used atomic force microscopy (AFM), a high resolution imaging technique. The first chapter is devoted to explain the basic concepts of AFM technique, as well as the composition and structure of DNA molecules. We also describe some of the polymer physics models of DNA, defining basic quantities like the persistence and the critical exponent. In chapter 2 we discuss experiments, in which we studied changes in the physical properties of DNA molecules, when bound to the substrate surface with various methods. We show that, when DNA is strongly bound to the surface, it retains its physiological B-form. On contrary, when weakly bound, a conformational B-to-A-form transition takes place. We also discuss a novel experimental method, combining nanometer sized PDMS slits with AFM, for studying DNA in geometrical confinement. When DNA is weakly confined, DNA persistence length increases, molecules elongate and adopt more anisotropic shapes. Under stronger confinement, DNA hairpins form. In chapter 3 we study binding of staining dyes, proteins and RNA polymerase (RNAP) to DNA molecules. First we show that DNA physical properties are significantly affected when stained with dyes. Depending on the dye binding mode, either the contour length, or the persistence length or both simultaneously change. We move further and investigate the role of protein amino acids and DNA sequence in the formation of nucleoprotein complexes. On the example of EspR protein, a key virulence regulator in Mycobacterium tuberculosis (MTB), we show that single amino acid mutations, lead to lower DNA binding affinity. These mutations also impair the formation of higher order protein-DNA complexes, silencing the MTB virulence. Afterwards, on the examples of RNAP and H-NS protein, we show the importance of DNA sequence for the binding and higher order oligomerization. By introducing DNA mutations disrupting the helical phasing between RNAP binding sites in the fis promoter sequence, we significantly lower the RNAP binding affinity. The helical phasing of the binding sites somehow coordinates the cooperative binding of RNAP to the promoter. Finally on the model of H-NS protein, we show how different spatial arrangements of strong binding sites for an architectural protein in the DNA, determine the final 3D structure of the assembled nucleoprotein complex. The final chapter, is fully devoted to the study of a completely new type of DNA organisation, which we call Hyperplectonemes. Hyperplectonemes are very ordered DNA structures, formed by large supercoiled molecules, in the presence of attractive DNA-DNA potential. First, we describe their structure and its dependence on various environmental factors, than their binding to nucleoid associated proteins. We argue that this emerging DNA organisation is the basic structure of the bacterial chromatin, which is further modulated by numerous DNA binding and condensing proteins in vivo.

EPFL2015

Molecular Mechanisms Underlying Hyaline Fibromatosis Syndrome

Julie Deuquet Ariosa

Hyaline Fibromatosis Syndrome (HFS) is a rare inherited disease that is characterized by an accumulation of an unidentified hyaline material, largely affecting connective tissues. Patients afflicted with HFS present a wide range of clinical symptoms such as nodules, papules, hyperplasia of the gingiva, joint contractions, thickness and hyperpigmentation of the skin, and osteopenia. Depending of the severity and the delay of appearance of these symptoms, the life expectancy varies between 2 years and > 30 years. Death earlier in life is due to recurrent diarrhea and chest infections of unknown origin. In 2003, the gene responsible for HFS was identified to be Capillary Morphoqenesis Gene 2 (CMG2), a gene upregulated during capillary morphogenesis in three-dimensional collagen matrices. This gene encodes a type I transmembrane glycoprotein composed of a von Willebrand (vWA) domain followed by an immunoglobulin (Ig)-like domain in its extracellular part, a single α-helix spanning transmembrane domain, and a 148 amino acids cytoplasmic tail predicted as unstructured. Even though the physiological function of CMG2 remains to be elucidated, it has been shown that this protein induces endothelial proliferation, and dictates cell polarization during embryogenesis in a zebrafish model. Besides its physiological function, CMG2 was found to be an anthrax toxin receptor, thus its alternative name ANTXR2, and has been extensively studied in this context. Genetic analysis has led to the identification of 34 CMG2 mutations in HFS patients. The molecular mechanisms underlying the disease have however not yet been identified. During the five years of my thesis, we were interested in understanding the cellular consequences of these mutations and in getting a better understanding of the physiological function of CMG2. I first showed that most of the HFS mutations in the vWA domain lead to alterations in folding of the domain, either in terms of kinetics or of structure, leading to recognition of the protein by the ER quality control (ERQC) and subsequent degradation by the ERAD pathway. A direct consequence of this retention is the loss of function at the cell surface or other cellular sites. In a second study, I studied the effect of novel HFS mutations that our collaborators and we identified in the CMG2 Ig-like domain. Since the structure of this domain was uncharacterized, we first generated a model of the domain and found that it has an Ig-like fold, despite the very low sequence homology, and harbors two disulfide bonds essential for folding and stabilization of the domain. HFS mutations localized to this domain led to aberrant intermolecular disulfide binding, ER retention and enhanced ERAD when compared to the vWA domain HFS mutations as shown in patients fibroblasts. The consequential loss of CMG2 could be partly alleviated by treating cells with proteasome inhibitor, such as Bortezomib, illustrating that ERAD components are potential therapeutic targets for HFS. As most of the HFS mutations in the ectodomain of CMG2 trigger this ER retention during the CMG2 biosynthesis, we then addressed this process by studying one of the most described post-translational modifications occurring in the ER for protein folding, namely the N-glycosylation. We found that the two potential CMG2 N-glycosylation sites, N250 and N260, are both occupied. Neither of the two sites is essential for ER exit. Glycosylation of CMG2 on N260 is however important since the expression levels of the N260A mutant were significant lower that for the WT protein, despite equivalent or higher synthesis, suggesting that glycosylation at this site is important for efficient folding. Finally, in order to better understand the CMG2 function, we investigated what the physiological ligands could be, assuming it is a receptor as suggested by its role in anthrax infection. We identified type VI collagen as a potential ligand in-vitro that activates CMG2 in-vivo, by phosphorylation of CMG2 cytoplasmic tail. This CMG2 activation upon type VI collagen binding could be a signal for ligand-triggered endocytosis, as is has been described with the protective antigen (PA) of anthrax toxin. The results of this work allow us to gain insights in the molecular mechanisms underlying HFS. Our studies indicate that the identification of the genotype-phenotype correlation of this disease is important for proper diagnosis and clinical approaches, and also that a therapeutic strategy based on proteasome inhibitors, such as Bortezomib, could be envisioned to treat some HFS symptoms in order to improve the life conditions of the patients.

EPFL2011

Identification of a Dps contamination in Mitomycin-C–induced expression of Colicin Ia

Henning Paul-Julius Stahlberg

Colicins are bacterial toxins targeting Gram-negative bacteria, including E. coli and related Enterobacteriaceae strains. Some colicins form ion-gated pores in the inner membrane of attacked bacteria that are lethal to their target. Colicin Ia was the first pore-forming E. coli toxin, for which a high-resolution structure of the monomeric full-length protein was determined. It is so far also the only colicin, for which a low-resolution structure of its membrane-inserted pore was reported by negative-stain electron microscopy. Resolving this structure at the atomic level would allow an understanding of the mechanism of toxin pore formation. Here, we report an observation that we made during an attempt to determine the Colicin Ia pore structure at atomic resolution. Colicin Ia was natively expressed by mitomycin-C induction under a native SOS promotor and purified following published protocols. The visual appearance in the electron microscope of negatively stained preparations and the lattice parameters of 2D crystals obtained from the material were highly similar to those reported earlier resulting from the same purification protocol. However, a higher-resolution structural analysis revealed that the protein is Dps (DNA-binding protein from starved cells), a dodecameric E. coli protein. This finding suggests that the previously reported low-resolution structure of a “Colicin Ia oligomeric pore” actually shows Dps.