Invadolysin is a novel metalloprotease conserved amongst metazoans that is essential for life in
. We previously showed that invadolysin was essential for the cell cycle and cell migration, linking ...to metabolism through a role in lipid storage and interaction with mitochondrial proteins. In this study we demonstrate that
mutants exhibit increased autophagy and decreased glycogen storage – suggestive of a role for invadolysin in insulin signaling in
. Consistent with this, effectors of insulin signaling were decreased in
mutants. In addition, we discovered that invadolysin was deposited on newly synthesized lipid droplets in a PKC-dependent manner. We examined two
models of adipogenesis for the expression and localization of invadolysin. The level of invadolysin increased during both murine 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS), adipogenesis. Invadolysin displayed a dynamic localization to lipid droplets over the course of adipogenesis, which may be due to the differential expression of distinct invadolysin variants. Pharmacological inhibition of adipogenesis abrogated the increase in invadolysin. In summary, our results on
and
systems highlight an important role for invadolysin in insulin signaling and adipogenesis.
We identified invadolysin, a novel essential metalloprotease, for functions in chromosome structure, cell proliferation and migration. Invadolysin also plays an important metabolic role in insulin ...signalling and is the only protease known to localise to lipid droplets, the main lipid storage organelle in the cell.
examination of the protein sequence of invadolysin predicts not only protease and lipase catalytic motifs, but also post-translational modifications and the secretion of invadolysin. Here we show that the protease motif of invadolysin is important for its role in lipid accumulation, but not in glycogen accumulation. The lipase motif does not appear to be functionally important for the accumulation of lipids or glycogen. Post-translational modifications likely contribute to modulating the level, localisation or activity of invadolysin. We identified a secreted form of invadolysin in the soluble fraction of invertebrate hemolymph (where we observe sexually dimorphic forms) and also vertebrate plasma, including in the extracellular vesicle fraction. Biochemical analysis for various post-translational modifications demonstrated that secreted invadolysin is both N- and O-glycosylated, but not apparently GPI-linked. The discovery of invadolysin in the extracellular milieu suggests a role for invadolysin in normal organismal physiology.
Emerin and lamin B receptor (LBR) are abundant transmembrane proteins of the nuclear envelope that are concentrated at the inner nuclear membrane (INM). Although both proteins interact with chromatin ...and nuclear lamins, they have distinctive biochemical and functional properties. Here, we have deployed proximity labeling using the engineered biotin ligase TurboID (TbID) and quantitative proteomics to compare the neighborhoods of emerin and LBR in cultured mouse embryonic fibroblasts. Our analysis revealed 232 high confidence proximity partners that interact selectively with emerin and/or LBR, 49 of which are shared by both. These included previously characterized NE-concentrated proteins, as well as a host of additional proteins not previously linked to emerin or LBR functions. Many of these are TM proteins of the ER, including two E3 ubiquitin ligases. Supporting these results, we found that 11/12 representative proximity relationships identified by TbID also were detected at the NE with the proximity ligation assay. Overall, this work presents methodology that may be used for large-scale mapping of the landscape of the INM and reveals a group of new proteins with potential functional connections to emerin and LBR.
Invadolysin is a novel metalloprotease, which is conserved amongst metazoans and was first identified in the Heck laboratory. Proteases play a variety of roles in normal physiology. Invadolysin is ...essential for life in Drosophila. Invadolysin has been shown to be essential for cell division and cell migration. Invadolysin is the only metalloprotease that we know of which localizes to lipid droplets, the lipid storage cell organelle. Previous studies have also shown that invadolysin mutants have a lower triglyceride to protein ratio and reduced fat body thickness and cross sectional area. Fat body in Drosophila is the functionally homolog of adipose tissue in higher organisms. Further suggesting a role of invadolysin in metabolism. In the Heck laboratory, invadolysin is studied using model organisms such as Drosophila melanogaster, Danio rerio (zebrafish) and cultured cell lines. During my PhD, my aim was to study the biosynthesis, activity and function of invadolysin and investigate its role in metabolism and adipogenesis. Invadolysin has a conserved metalloprotease motif ‘HEXXH’ and a potential lipase motif ‘GXSXS’. One of the aims of my PhD was to generate mutant versions of the conserved motifs to study their role on the activity of the proteins. I have generated transgenic flies that express wild type or E258A (protease dead) or S266A (lipase dead) versions of invadolysin. These transgenic flies would help in the study of the importance of the metalloprotease ‘HEILH’ and the lipase ‘GFSVS’ motifs in invadolysin’s activity. Transgenic flies overexpressing wild type and lipase dead form of invadolysin accumulate significantly higher levels of triglycerides as compared to control flies and transgenic flies overexpressing protease dead form of invadolysin. Suggesting a role of the protease motif in lipid accumulation. The other aim of my PhD was to study the role of invadolysin in metabolism. I followed up on previous observations in the laboratory that the insulin-signalling pathway is impaired in invadolysin mutant animals – with the hypothesis that invadolysin plays a role in metabolism and adipogenesis. I used Drosophila to study the effect on downstream targets of the insulin-signalling pathway such as triglyceride synthesis, glycogen synthesis and autophagy in invadolysin mutants. Results suggest that the insulin-signalling pathway and the ability to accumulate lipids are impaired in invadolysin mutants. Insulin also regulates adipogenesis by regulating the expression of PPARγ. I used SGBS cells, a human preadipocyte cell line to study the role of invadolysin in adipogenesis. Increase in protein levels of invadolysin during adipogenesis indicates a potential role of invadolysin in adipogenesis. Invadolysin has a predicted N-terminal signal sequence and also a predicted Cterminus GPI anchor site that suggests invadolysin can either be secreted or anchored to a membrane. Also, leishmanolysin, the closest homolog of invadolysin exists in a secreted and membrane bound form apart from a cytosolic form. This encouraged me to investigate the presence of a secreted form of invadolysin. Analysis of vertebrate and invertebrate plasma fractions of blood and hemolymph led to the identification of a novel secreted form of invadolysin. This novel discovery places invadolysin alongside a small group of metalloproteases, which are secreted into the extracellular environment and which play multiple roles in normal physiology and disease states.
Identification of components essential to chromosome structure and behaviour remains a vibrant area of study. We have previously shown that invadolysin is essential in Drosophila, with roles in cell ...division and cell migration. Mitotic chromosomes are hypercondensed in length, but display an aberrant fuzzy appearance. We additionally demonstrated that in human cells, invadolysin is localized on the surface of lipid droplets, organelles that store not only triglycerides and sterols but also free histones H2A, H2Av and H2B. Is there a link between the storage of histones in lipid droplets and the aberrantly structured chromosomes of invadolysin mutants? We have identified a genetic interaction between invadolysin and nonstop, the de-ubiquitinating protease component of the SAGA (Spt-Ada-Gcn5-acetyltransferase) chromatin-remodelling complex. invadolysin and nonstop mutants exhibit phenotypic similarities in terms of chromosome structure in both diploid and polyploid cells. Furthermore, IX-14(1)/not(1) transheterozygous animals accumulate mono-ubiquitinated histone H2B (ubH2B) and histone H3 tri-methylated at lysine 4 (H3K4me3). Whole mount immunostaining of IX-14(1)/not(1) transheterozygous salivary glands revealed that ubH2B accumulates surprisingly in the cytoplasm, rather than the nucleus. Over-expression of the Bre1 ubiquitin ligase phenocopies the effects of mutating either the invadolysin or nonstop genes. Intriguingly, nonstop and mutants of other SAGA subunits (gcn5, ada2b and sgf11) all suppress an invadolysin-induced rough eye phenotype. We conclude that the abnormal chromosome phenotype of invadolysin mutants is likely the result of disrupting the histone modification cycle, as accumulation of ubH2B and H3K4me3 is observed. We further suggest that the mislocalization of ubH2B to the cytoplasm has additional consequences on downstream components essential for chromosome behaviour. We therefore propose that invadolysin plays a crucial role in chromosome organization via its interaction with the SAGA complex.
Invadolysin is a novel metalloprotease, which is conserved amongst metazoans and was first identified in the Heck laboratory. Proteases play a variety of roles in normal physiology. Invadolysin is ...essential for life in Drosophila. Invadolysin has been shown to be essential for cell division and cell migration. Invadolysin is the only metalloprotease that we know of which localizes to lipid droplets, the lipid storage cell organelle. Previous studies have also shown that invadolysin mutants have a lower triglyceride to protein ratio and reduced fat body thickness and cross sectional area. Fat body in Drosophila is the functionally homolog of adipose tissue in higher organisms. Further suggesting a role of invadolysin in metabolism. In the Heck laboratory, invadolysin is studied using model organisms such as Drosophila melanogaster, Danio rerio (zebrafish) and cultured cell lines. During my PhD, my aim was to study the biosynthesis, activity and function of invadolysin and investigate its role in metabolism and adipogenesis. Invadolysin has a conserved metalloprotease motif ‘HEXXH’ and a potential lipase motif ‘GXSXS’. One of the aims of my PhD was to generate mutant versions of the conserved motifs to study their role on the activity of the proteins. I have generated transgenic flies that express wild type or E258A (protease dead) or S266A (lipase dead) versions of invadolysin. These transgenic flies would help in the study of the importance of the metalloprotease ‘HEILH’ and the lipase ‘GFSVS’ motifs in invadolysin’s activity. Transgenic flies overexpressing wild type and lipase dead form of invadolysin accumulate significantly higher levels of triglycerides as compared to control flies and transgenic flies overexpressing protease dead form of invadolysin. Suggesting a role of the protease motif in lipid accumulation. The other aim of my PhD was to study the role of invadolysin in metabolism. I followed up on previous observations in the laboratory that the insulin-signalling pathway is impaired in invadolysin mutant animals – with the hypothesis that invadolysin plays a role in metabolism and adipogenesis. I used Drosophila to study the effect on downstream targets of the insulin-signalling pathway such as triglyceride synthesis, glycogen synthesis and autophagy in invadolysin mutants. Results suggest that the insulin-signalling pathway and the ability to accumulate lipids are impaired in invadolysin mutants. Insulin also regulates adipogenesis by regulating the expression of PPARγ. I used SGBS cells, a human preadipocyte cell line to study the role of invadolysin in adipogenesis. Increase in protein levels of invadolysin during adipogenesis indicates a potential role of invadolysin in adipogenesis. Invadolysin has a predicted N-terminal signal sequence and also a predicted Cterminus GPI anchor site that suggests invadolysin can either be secreted or anchored to a membrane. Also, leishmanolysin, the closest homolog of invadolysin exists in a secreted and membrane bound form apart from a cytosolic form. This encouraged me to investigate the presence of a secreted form of invadolysin. Analysis of vertebrate and invertebrate plasma fractions of blood and hemolymph led to the identification of a novel secreted form of invadolysin. This novel discovery places invadolysin alongside a small group of metalloproteases, which are secreted into the extracellular environment and which play multiple roles in normal physiology and disease states.
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