Identification of two novel human neuronal ceroid lipofuscinosis genes

The neuronal ceroid lipofuscinoses (NCLs) are a group of mostly autosomal recessively inherited neurodegenerative disorders. The aim of this thesis was to characterize the molecular genetic bases of these, previously genetically undetermined, NCL forms. Congenital NCL is the most aggressive form of NCLs. Previously, a mutation in the cathepsin D (CTSD) gene was shown to cause congenital NCL in sheep. Based on the close resemblance of the phenotypes between congenital NCLs in sheep and human, CTSD was considered as a potential candidate gene in humans as well. When screened for mutations by sequencing, a homozygous nucleotide duplication creating a premature stop codon was identified in CTSD in one family with congenital NCL. While in vitro the overexpressed truncated mutant protein was stable although inactive, the absence of CTSD staining in brain tissue samples of patients indicated degradation of the mutant CTSD in vivo. A lack of CTSD staining was detected also in another, unrelated family with congenital NCL. These results imply that CTSD deficiency underlies congenital NCL. While initially Turkish vLINCL was considered a distinct genetic entity (CLN7), mutations in the CLN8 gene were later reported to account for the disease in a subset of Turkish patients with vLINCL. To further dissect the genetic basis of the disease, all known NCL genes were screened for homozygosity by haplotype analysis of microsatellite markers and/or sequenced in 13 mainly consanguineous, Turkish vLINCL families. Two novel, family-specific homozygous mutations were identified in the CLN6 gene. In the remaining families, all known NCL loci were excluded. To identify novel gene(s) underlying vLINCL, a genomewide single nucleotide polymorphism scan, homozygosity mapping, and positional candidate gene sequencing were performed in ten of these families. On chromosome 4q28.1-q28.2, a novel major facilitator superfamily domain containing 8 (MFSD8) gene with six family-specific homozygous mutations in vLINCL patients was identified. MFSD8 transcript was shown to be ubiquitously expressed with a complex pattern of alternative splicing. Our results suggest that MFSD8 is a novel lysosomal integral membrane protein which, as a member of the major facilitator superfamily, is predicted to function as a transporter. Identification of MFSD8 emphasizes the genetic heterogeneity of Turkish vLINCL. In families where no MFSD8 mutations were detected, additional NCL-causing genes remain to be identified. The identification of CTSD and MFSD8 increases the number of known human NCL-causing genes to eight, and is an important step towards the complete understanding of the genetic spectrum underlying NCLs. In addition, it is a starting point for dissecting the molecular mechanisms behind the associated NCLs and contributes to the challenging task of understanding the molecular pathology underlying the group of NCL disorders.

Bending the Rules of Cell Protrusions : Molecular Mechanisms and Biological Roles of Inverse-BAR Proteins in Cell Morphogenesis

Plasma membrane adopts myriad of different shapes to carry out essential cellular processes such as nutrient uptake, immunological defence mechanisms and cell migration. Therefore, the details how different plasma membrane structures are made and remodelled are of the upmost importance. Bending of plasma membrane into different shapes requires substantial amount of force, which can be provided by the actin cytoskeleton, however, the molecules that regulate the interplay between the actin cytoskeleton and plasma membrane have remained elusive. Recent findings have placed new types of effectors at sites of plasma membrane remodelling, including BAR proteins, which can directly bind and deform plasma membrane into different shapes. In addition to their membrane-bending abilities, BAR proteins also harbor protein domains that intimately link them to the actin cytoskeleton. The ancient BAR domain fold has evolved into at least three structurally and functionally different sub-groups: the BAR, F-BAR and I-BAR domains. This thesis work describes the discovery and functional characterization of the Inverse-BAR domains (I-BARs). Using synthetic model membranes, we have shown that I-BAR domains bind and deform membranes into tubular structures through a binding-surface composed of positively charged amino acids. Importantly, the membrane-binding surface of I-BAR domains displays an inverse geometry to that of the BAR and F-BAR domains, and these structural differences explain why I-BAR domains induce cell protrusions whereas BAR and most F-BAR domains induce cell invaginations. In addition, our results indicate that the binding of I-BAR domains to membranes can alter the spatial organization of phosphoinositides within membranes. Intriguingly, we also found that some I-BAR domains can insert helical motifs into the membrane bilayer, which has important consequences for their membrane binding/bending functions. In mammals there are five I-BAR domain containing proteins. Cell biological studies on ABBA revealed that it is highly expressed in radial glial cells during the development of the central nervous system and plays an important role in the extension process of radial glia-like C6R cells by regulating lamellipodial dynamics through its I-BAR domain. To reveal the role of these proteins in the context of animals, we analyzed MIM knockout mice and found that MIM is required for proper renal functions in adult mice. MIM deficient mice displayed a severe urine concentration defect due to defective intercellular junctions of the kidney epithelia. Consistently, MIM localized to adherens junctions in cultured kidney epithelial cells, where it promoted actin assembly through its I-BAR andWH2 domains. In summary, this thesis describes the mechanism how I-BAR proteins deform membranes and provides information about the biological role of these proteins, which to our knowledge are the first proteins that have been shown to directly deform plasma membrane to make cell protrusions.

Structure-Function Studies of GDNF Family Ligand-RET signalling

Glial cell line-derived neurotrophic factor (GDNF) and its family members neurturin (NRTN), artemin (ARTN) and persephin (PSPN) are growth factors, which are involved in the development, differentiation and maintenance of many neuron types. In addition, they function outside of the nervous system, e.g. in the development of kidney, testis and liver. GDNF family ligand (GFL) signalling happens through a tetrameric receptor complex, which includes two glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor (GFRα) molecules and two RET (rearranged during transfection) receptor tyrosine kinases. Each of the ligands binds preferentially one of the four GFRα receptors: GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The signal is then delivered by RET, which cannot bind the GFLs on its own, but can bind the GFL-GFRα complex. Under normal cellular conditions, RET is only phosphorylated on the cell surface after ligand binding. At least the GDNF-GFRα1 complex is believed to recruit RET to lipid rafts, where downstream signalling occurs. In general, GFRαs consist of three cysteine-rich domains, but all GFRα4s except for chicken GFRα4 lack domain 1 (D1). We characterised the biochemical and cell biological properties of mouse PSPN receptor GFRα4 and showed that it has a significantly weaker capacity than GFRα1 to recruit RET to the lipid rafts. In spite of that, it can phosphorylate RET in the presence of PSPN and contribute to neuronal differentiation and survival. Therefore, the recruitment of RET to the lipid rafts does not seem to be crucial for the biological activity of all GFRα receptors. Secondly, we demonstrated that GFRα1 D1 stabilises the GDNF-GFRα1 complex and thus affects the phosphorylation of RET and contributes to the biological activity. This may be important in physiological conditions, where the concentration of the ligand or the soluble GFRα1 receptor is low. Our results also suggest a role for D1 in heparin binding and, consequently, in the biodistribution of released GFRα1 or in the formation of the GFL-GFRα-RET complex. We also presented the crystallographic structure of GDNF in the complex with GFRα1 domains 2 and 3. The structure differs from the previously published ARTN-GFRα3 structure in three significant ways. The biochemical data verify the structure and reveal residues participating in the interactions between GFRα1 and GDNF, and preliminarily also between GFRα1 and RET and heparin. Finally, we showed that, the precursor of the oncogenic MEN 2B (multiple endocrine neoplasia type 2) form of RET gets phosphorylated already during its synthesis in the endoplasmic reticulum (ER). We also demonstrated that it associates with Src homology 2 domain-containing protein (SHC) and growth factor receptor-bound protein (GRB2) in the ER, and has the capacity to activate several downstream signalling molecules.

Functions of Alphavirus Macrodomain-containing protein nsP3

Alphaviruses are positive strand RNA viruses that replicate in association with cellular membranes. The viral RNA replication complex consists of four non-structural proteins nsP1-nsP4 which are essential for viral replication. The functions of nsP1, nsP2 and nsP4 are well established, but the roles of nsP3 are mainly unknown. In this work I have clarified some of the functions of nsP3 in order to better understand the importance of this protein in virus replication. Semliki Forest virus (SFV) has been mostly used as a model alphavirus during this work, but some experiments have also been conducted with Sindbis and Chikungunya viruses. NsP3 is composed of three different protein domains. The N-terminus of nsP3 contains an evolutionarily conserved macrodomain, the central part of nsP3 contains a domain that is only found in alphaviruses, and the C-terminus of the protein is hypervariable and predicted to be unstructured. In this work I have analyzed the functions of nsP3 macrodomain, and shown that viral macrodomains bind poly(ADP-ribose) and that they do not resemble cellular macrodomains in their properties. Furthermore, I have shown that some macrodomains, including viral macrodomains of SFV and hepatitis E virus, also bind poly(A). Mutations in the ligand binding pocket of SFV macrodomain hamper virus replication but do not confer lethality, indicating that macrodomain function is beneficial but not mandatory for virus replication. The hypervariable C-terminus of nsP3 is heavily phosphorylated and is enriched in proline residues. In this work it is shown that this region harbors an SH3 domain binding motif (Sh3BM) PxRxPR through which cellular amphiphysin is recruited to viral replication sites and to nsP3 containing cytoplasmic aggregate structures. The function of Sh3BM was destroyed by a single point mutation, which led to impaired viral RNA replication in HeLa cells, pointing out the functional importance of amphiphysin recruitment by the Sh3BM. In addition, evidence is provided tho show that the endosomal localization of alphavirus replication is mediated by nsP3 and that the phosphorylation of hypervariable region might be important for the endosomal targeting. Together these findings demonstrate that nsP3 contains multiple important host interaction motifs and domains, which facilitate successful viral propagation in host cells.