The interaction between LC8 and LCA5 reveals a novel oligomerization function of LC8 in the ciliary-centrosome system

Dynein light chain LC8 is a small dimeric hub protein that recognizes its partners through short linear motifs and is commonly assumed to drive their dimerization. It has more than 100 known binding partners involved in a wide range of cellular processes. Recent large-scale interaction studies suggested that LC8 could also play a role in the ciliary/centrosome system. However, the cellular function of LC8 in this system remains elusive. In this work, we characterized the interaction of LC8 with the centrosomal protein lebercilin (LCA5), which is associated with a specific form of ciliopathy. We showed that LCA5 binds LC8 through two linear motifs. In contrast to the commonly accepted model, LCA5 forms dimers through extensive coiled coil formation in a LC8-independent manner. However, LC8 enhances the oligomerization ability of LCA5 that requires a finely balanced interplay of coiled coil segments and both binding motifs. Based on our results, we propose that LC8 acts as an oligomerization engine that is responsible for the higher order oligomer formation of LCA5. As LCA5 shares several common features with other centrosomal proteins, the presented LC8 driven oligomerization could be widespread among centrosomal proteins, highlighting an important novel cellular function of LC8

The Role of OFD1 in the Pathogenesis of Polycystic Kidney Disease

Oral-facial-digital syndrome type1 (OFD1) is an inherited disorder caused by expression of mutant OFD1 protein and results in abnormalities in facial and limb development and polycystic kidney disease that resembles Autosomal Dominant Polycystic Kidney Disease (ADPKD). The similarities in renal disease pathogenesis in patients with OFD1 and ADPKD suggest that the proteins implicated in these diseases, OFD1 and the polycystins, respectively, function on a common pathway in the affected cell types. This calls for an investigation to uncover the mechanism by which OFD1 contributes to polycystic kidney disease pathogenesis, which remains unknown. This thesis details for the first time the assembly of a ciliary signaling microdomain containing OFD1, the polycystins, the epidermal growth factor receptor (EGFR), and the domain organizing flotillin proteins in renal epithelia and cells of the oral cavity. Also, the studies presented here provide a mechanism by which OFD1, a soluble protein, is trafficked to primary cilia via its interaction with polycystin-1 (PC1), a ciliary membrane protein. The presented data demonstrate the requirement of coiled coil domains found in both OFD1 and PC1 for OFD1 interaction with PC1 and proper localization of OFD1 to primary cilia. The composite work provides the foundation for identifying potential molecular targets for therapeutic interventions for the millions of individuals suffering from polycystic kidney disease worldwide

Elucidating the function of WAC in macroautophagy through investigating the WAC-GM130 interaction

Macroautophagy, hereafter called autophagy, is an intracellular degradation pathway that functions to degrade bulk material and recycle macromolecules to maintain cell homeostasis. Autophagy has a protective role and is also implicated in disease processes such as cancer, neurodegeneration and pathogen clearance. Autophagy is characterised by the formation of a double-membrane structure in response to stress insults such as amino acid starvation, which encapsulates cargo to form an enlarged vesicle called an autophagosome. The autophagosome fuses with the lysosome for degradation of the contents. Although the core protein machinery of autophagy has been known for some time, the regulation of this process is not fully understood. WAC was previously identified as a novel positive regulator of starvation-induced autophagy. However the mechanism by which WAC regulates autophagy is completely unknown. It is known that WAC performs functions both in the nucleus and on the Golgi. In order to elucidate the function of WAC in autophagy, I performed a study of WAC function by a combination of microarray transcriptomic analysis and immunoprecipitation coupled with mass spectrometry to identify genes regulated by WAC and novel WAC interaction partners, respectively. WAC is not a potent regulator of autophagy gene expression. However, I identified GM130, a negative regulator of autophagy, as a novel WAC interaction partner. I show here that WAC promotes ULK1 activation whereas GM130 inhibits the early autophagy stages. GM130 is the receptor for WAC on the Golgi and binds WAC directly. This interaction is mediated by the C-terminal regions of both proteins which contain predicted coiled-coil domains. WAC and GM130 both interact with the autophagy protein GABARAP, indirectly and directly respectively. WAC regulates a novel centrosomally-localised pool of GABARAP, GABARAP binding to GM130 and GABARAP localisation to the Golgi. This centrosomal pool of GABARAP, is able to translocate to autophagic structures during starvation. In addition I describe a GABARAP mediated activation of the ULK1 complex. Through the WAC-GM130-GABARAP interplay, I propose that WAC promotes a centrosome to autophagosome GABARAP trafficking step that may in addition maintain ULK1 activation during starvation-induced autophagy

Characterising novel mitotic microtubule associated proteins in the early Drosophila embryo

Cell division is a fundamental biological process driven by the formation of a microtubule (MT) based mitotic spindle, ensuring the accurate segregation of chromosomes. MT length, nucleation and dynamics are all determined by microtubule associated proteins (MAPs). Following on from previous work carried out by the Hughes et al., (2008) this investigation has applied quantitative comparative proteomics to cycling and mitotic Drosophila embryo extracts prepared via MT cosedimentation assay, in order to compare their MAP complements as they progress though the cell cycle or whilst fixed at the metaphase-anaphase transition. We have demonstrated that many MAPs known for their roles in cell division increase their association with MTs during mitosis, and in addition our approach has identified a number of protein classes not previously characterised as a MAP, binding to MTs during mitosis. One of these protein classes was the Replication Factor C complex (RFC). The RFC complex is most well-known for its role as the sliding clamp loader in DNA replication, however it is now presenting as MAPs with a potential mitotic function. By combining techniques from biochemistry, fluorescence microscopy and further proteomic analysis we have been able to begin to investigate the localisation and functions of this complex during mitosis. Thus far we have been able to biochemically show that the RFC complex is a true MT binding protein and that all three alternative RFC complexes, as well as the archetypal complex, are present in mitotic embryo extracts following immunoprecipitation of RFC3. We have also shown via fluorescence imaging that the RFC complex presents a weak localisation to the mitotic spindle. Application of these techniques has also led to further investigation into the known MAPs, Asp and DTACC, for which we have identified novel protein interactors and investigated localisation during the process of Drosophila embryonic spindle self-assembly

Proteomic profile of maternal-aged blastocoel fluid suggests a novel role for ubiquitin system in blastocyst quality

Purpose: The etiology of maternal aging, a common cause of female factor infertility and a rate-limiting step in vitro fertilization (IVF) success, remains still unclear. Proteomic changes responsible for the impaired successful pregnancy outcome after IVF with aged blastocysts have not been yet evaluated. The objective of this prospective study was to employ proteomic techniques and bioinformatic tools to enlight differences at the protein level in blastocoel fluid of aged and younger woman. Methods: Protein composition of human blastocoel fluid isolated by micromanipulation from 46 blastocysts of women aged <37 years (group A) and 29 of women aged 6537 years (group B) have been identified by a shotgun proteomic approach based on high-resolution nano-liquid chromatography electrospray-ionization-tandem mass spectrometry (nLC-ESI-MS/MS) using label free for the relative quantification of their expression levels. Results: The proteomic analysis leads to the identification and quantification of 148 proteins; 132 and 116 proteins were identified in groups A and B, respectively. Interestingly, the identified proteins are mainly involved in processes aimed at fine tuning embryo implantation and development. Among the 100 proteins commonly expressed in both groups, 17 proteins are upregulated and 44 downregulated in group B compared to group A. Overall, the analysis identified 33 proteins, which were increased or present only in B while 76 were decreased in B or present only in A. Conclusions: Data revealed that maternal aging mainly affects blastocyst survival and implantation through unbalancing the equilibrium of the ubiquitin system known to play a crucial role in fine-tuning several aspects required to ensure successful pregnancy outcome

The Role of Trim58 in Erythropoiesis

Red blood cells (erythrocytes) deliver oxygen to all tissues of the body. Defects in red blood cell production (erythropoiesis) can cause disease. Mammalian erythropoiesis culminates in enucleation, an incompletely understood process that entails the physical separation of the nucleus and cytoplasm. The work in this thesis investigated the role of a previously uncharacterized protein named Trim58 in erythropoiesis. Human genetic studies suggested that TRIM58 played an important role in erythroid development. In humans and mice, Trim58 expression was found to be restricted to red blood cell precursors during late stage maturation. In fact, murine Trim58 was upregulated just prior to enucleation. Using short hairpin RNAs, Trim58 expression was inhibited in cultured murine erythroblasts. Through a variety of analyses, it was demonstrated that Trim58 is dispensable for early erythroid maturation. However, Trim58 knockdown impaired movement of the nucleus, thereby inhibiting enucleation. Trim58 is a member of the tripartite motif-containing family of proteins, many of which function as E3 ubiquitin ligases that can facilitate protein degradation. Protein interaction studies demonstrated that Trim58 bound directly to the molecular motor protein complex dynein. Consistent with its putative role as an E3 ubiquitin ligase, ectopic Trim58 expression in HeLa cells caused dynein degradation in a proteasome-dependent fashion. Furthermore, dynein loss and efficient enucleation were coincident and dependent upon Trim58 induction during erythroid culture maturation. Dynein mediates unidirectional nuclear movement toward the microtubule organizing center. Erythroid enucleation requires nuclear movement in the opposite direction. Hence, Trim58-mediated dynein degradation may be responsible for nuclear movement during enucleation. Our findings identify Trim58 as the first erythroid-specific protein that regulates this process. More broadly, regulated proteolysis represents a previously unappreciated mode of regulation for dynein, which is critical for many cellular processes