Invadolysin (INV) is a member of the M8 family of metzincin metalloproteases. The gene was discovered in the Heck laboratory. Based on studies in Drosophila, INV is important for mitotic progression, nuclear envelope protein dynamics, and germ cell migration. INV-like immunoreactivity has shown its association with lipid droplets (LDs), which are intracellular organelles for lipid and protein storage. INV is the first metalloprotease found on LDs. Thus, INV’s role and LD-associated pathways are the puzzles we would like to investigate. The formation of LDs is dependent on the nutritional status of cells and starvation can disrupt the generation of LDs. Based on this concept, I established a starvation / re-feeding system. When nutrition is sufficient, LDs were surrounded by INV, whereas no INV or LDs were found in the majority of starved cells. With a supply of oleic acid (OA), LDs re-appeared and so did INV localized to LDs. In this system, inhibition of protein kinase C (PKC) disrupts INV’s re-localization to LDs. As I found INV to be phosphorylated by PKC in vitro (residues within the N-terminus might be phosphorylated by PKC), I conclude that PKC might regulate INV’s re-localization in the starvation / re-feeding system. 3T3-L1 mouse fibroblasts can differentiate into adipocytes in vitro; this is termed adipogenesis. Since INV is a LD associated protein, the role of INV in adipogenesis is of interest. INV localized on LDs in the early stage of differentiation but disassociated from LDs in mature adipocytes. The levels of INV mRNA and protein were significantly increased upon differentiation to adipocytes. On the other hand, INV decreased when adipocyte differentiation was inhibited by PKC and PI3K inhibitors, suggesting that the increase of INV is required for adipocyte differentiation. I was interested to examine the possible role of INV in InR/PI3K/Akt signalling, and therefore compared wild type with mutant INV (Drosophila INV4Y7). Decreased levels of phospho-Akt and phospho-S6K, and increased mRNA levels of d4E-BP were observed in INV4Y7 mutant larvae, suggesting that INV may be required for InR/PI3K/Akt signalling. In addition, a decreased level of Lsd2 (LD binding protein) was found in INV4Y7 mutants. These correlations between INV and molecules important for signaling suggest that INV might be a mediator of nutritional metabolism. In light of these data, I speculate that INV plays a homeostatic role, possibly by affecting the InR/PI3K/Akt signaling pathway. In conclusion, the localization of INV to LDs is dependent on the activity of PKC. An increase in invadolysin accompanies adipogenesis, in which PKC and PI3K may be mediators. Examining mutant Drosophila, I found INV to be involved in InR/PI3K/Akt signalling. Collectively, I conclude that INV may serve as a regulator in adipogenesis and the InR/PI3K/Akt signaling pathway.

Coral reefs ecosystems are some of the most productive and diverse in the world. The symbiotic association between cnidarians and their symbiotic microalgae is of great importance in coral reef ecosystems; however, its underlying molecular mechanism remains unclear even after decades of research. Rab small GTP binding proteins are critical regulators of vesicle trafficking. Here we present the experimental evidence supporting a possible association of ApRab37 with the surface of lipid droplets in the endosymbiosis between the sea anemone, Aiptasia pulchella and the symbiotic dinoflagellate (commonly known as zooxanthellae). ApRab37, a protein of 215 amino acids, displays strong homology with human Rab37. In transfected COS7 cells, EGFP-ApRab37 localized to lipid droplets and clustered in the peri-nuclear region, which stained positive for the ER (endoplasmic reticulum) marker. Immunostaining analysis found ApRab37 associated with symbiosomes and lipid droplets, which was also confirmed by Western blot analysis of in the enrich lipid droplet fraction. Phagocytosis assay showed that ApRab37 involved in late phase of phagocytosis. DCMU treatment indicates symbiosome association of ApRab37 is mediated by zooxanthellae. I propose that ApRab37 plays a pivotal role in the regulation of lipid trafficking from the symbiosomes to the host cell during the Aiptasia-zooxanthellal endosymbiosis.

Indiana University-Purdue University Indianapolis (IUPUI) / Coxiella burnetii is an intracellular bacterium that causes the human disease Q fever. C. burnetii is transmitted from infected animals to humans through inhalation of infectious droplets. Acute Q fever is a flu-like illness lasting 10-14 days. Patients often have respiratory symptoms and present with pneumonia. Patients with suppressed immune systems or valvular heart disease can develop chronic Q fever, which causes endocarditis and vasculitis long after initial infection. Chronic Q fever is difficult to treat, and if untreated, is typically fatal. Currently, the United States lacks any vaccine for Q fever. In order to better prevent and treat this disease, it is important to understand how C. burnetii interacts with mammalian cells. Within the host cell, C. burnetii forms a large, acidic Coxiella-containing vacuole (CCV) and uses a Type 4B secretion system (T4SS) to secrete effector proteins into the host cell cytoplasm. While the CCV membrane is rich in sterols, cholesterol accumulation in the CCV is bacteriolytic, suggesting that C. burnetii regulation of lipid transport is critical for infection. The mammalian lipid transport protein ORP1L localizes to the CCV membrane and mediates CCV-ER membrane contact sites. ORP1L functions in lipid transport, including cholesterol efflux from late endosomes/lysosomes. Its sister isoform ORP1S binds cholesterol but localizes to the cytoplasm and nucleus. In ORP1- null cells, we found that CCVs were smaller than in wildtype cells, highlighting the importance of ORP1 in CCV development. CCVs in ORP1-null cells had higher cholesterol content than CCVs in wildtype cells, suggesting ORP1 functions in cholesterol efflux from the CCV. ORP1-null MH-S cells do not accumulate lipid droplets upon C. burnetii infection, supporting our hypothesis that ORP1 promotes cholesterol transfer from the CCV to the ER, as lipid droplets form from neutral lipids in the ER. While the absence of ORP1 led to a C. burnetii growth defect in MH-S cells, there was no growth defect in HeLa cells. Together, our data demonstrate that C. burnetii uses the host sterol transport protein ORP1 to promote CCV development, potentially by using ORP1 to facilitate cholesterol efflux from the CCV to diminish the bacteriolytic effects of cholesterol.

<p>Advances in modern medicine have helped to prolong human life. These advancements coupled with an ever-increasing population means that diseases associated with aging will become more prevalent in the coming years. As such, it is critical to understand the pathogenesis of disease where aging is the main risk factor. While not widely known, age is in fact a large risk factor in development of obesity and metabolic syndrome. More widely known and discussed are the neurodegenerative diseases that occur late in life. While age as a risk factor is a common point between these types of pathology, there are other similarities, such as the interaction between lipid metabolism and mitochondrial health. </p><p>To study the overlap between obesity and neurodegeneration, we investigated two pathways that regulate both. First, we find that loss of cytoplasmic deacetylase HDAC6 leads to aberrant accumulation of lipid in vitro and in vivo. HDAC6 knock-out (KO) mice gain more weight than WT counterparts after a high-fat diet regimen. Additionally, the intermediary metabolism of cells lacking HDAC6 is disrupted as they increase glucose uptake while downregulating fatty acid oxidation. HDAC6 not only plays a role in lipid metabolism, but regulates mitochondrial dynamics. Upon glucose-withdrawal, HDAC6 KO cells fail to elongate their mitochondria and display increased levels of mitochondrial toxic by-products. Therefore, HDAC6 has critical roles in lipid homeostasis and mitochondrial health. </p><p>The other pathway we investigated is critical in neurodegenerative disease, Parkinson's disease. Parkin, an E3 ubiquitin ligase, flags damaged mitochondria for destruction so they do not poison the other functional organelles. We found that Parkin promotes lipid remodeling at the surface of the mitochondria. Phosphatidic acid (PA) accumulates shortly after mitochondrial damage while diacylglycerol (DAG) appears several hours later. This lipid accumulation is dependent upon Parkin's translocation and E3 ligase activity. Additionally, we found that lipin-1, a PA phosphatase, and endophilin B1 (EndoB1) are critical for DAG accumulation and effective mitochondrial clearance. </p><p>Through this work, we show that two proteins critical in quality control mechanisms also play significant roles in energy homeostasis. We aim to highlight this overlap and posit that common diseases of aging, though presenting differently, might have disruptions in the same basic process.</p> / Dissertation

No description available.

Hepatitis C virus (HCV) is remarkably capable of efficiently hijacking host cell pathways including lipid metabolism in the liver in order to create pro-viral environments for pathogenesis. It is becoming increasingly clear that identifying small molecule inhibitors that target host factors exploited by the virus will expand available HCV treatment options. As such, a thorough understanding of host-virus interactions is critical to the development of alternative therapeutic strategies. Hepatic lipid droplets (LDs) are recruited by HCV to play essential roles in the viral lifecycle. The intracellular location of LDs is modified upon interacting with viral structural core protein. This enables formation of platforms that support viral particle assembly. Because these interactions are non-static, capturing its dynamic processes in order to better understand viral assembly can be achieved with label-free molecular imaging enhanced with live-cell capabilities. Chemical biology approaches that includes CARS microscopy employed in a multi-modal imaging system was used to probe interactions between HCV and host LDs. By successfully tracking LD trajectories, we identified core protein’s ability to alter LD speed and control for LD directionality. Using protein expression model systems that allowed for simultaneous tracking of core protein and LDs, our data revealed that mutations in the core protein region that vary in hydrophobicity and LD binding strengths, are factors that control for differential modulation of LD kinetics. Furthermore, we measured bidirectional LD travels runs and velocities, and observed critical properties by which core protein induces LD migration towards regions of viral particle assembly. Given that many steps in the HCV lifecycle are directly linked to host lipid metabolism, it is not surprising that disrupting lipid biosynthetic pathways would negatively affect viral replication. From this outlook, we explored small molecule inhibitors that targeted several lipid metabolic pathways to study its antiviral properties. Using fluorescent probes covalently labeled to viral RNA, we captured the visualization of disrupted replication complexes upon antagonizing nuclear hormone receptors that are linked to regulating lipid homeostasis. Correspondingly, biochemistry and molecular imaging techniques were also employed to identify novel antiviral mechanisms of small molecule inhibitors that target additional HCV-dependent lipid metabolic pathways.

No description available.

Improvements in mass spectrometry (MS)-based strategies for characterizing the plant lipidome through quantitative and qualitative approaches such as shotgun lipidomics have substantially enhanced our understanding of the structural diversity and functional complexity of plant lipids. However, most of these approaches require chemical extractions that result in the loss of the original spatial context and cellular compartmentation for these compounds. To address this current limitation, several technologies were developed to visualize lipids in situ with detailed chemical information. A subcellular visualization approach, direct organelle MS, was developed for directly sampling and analyzing the triacylglycerol contents within purified lipid droplets (LDs) at the level of a single LD. Sampling of single LDs demonstrated seed lipid droplet-to-droplet variability in triacylglycerol (TAG) composition suggesting that there may be substantial variation in the intracellular packaging process for neutral lipids in plant tissues. A cellular and tissue visualization approach, MS imaging, was implemented and enhanced for visualizing the lipid distributions in oilseeds. In mature cotton seed embryos distributions of storage lipids (TAGs) and their phosphatidylcholine (PCs) precursors were distribution heterogeneous between the cotyledons and embryonic axis raising new questions about extent and regulation of oilseed heterogeneity. Extension of this methodology provides an avenue for understanding metabolism in cellular (perhaps even subcellular) context with substantial metabolic engineering implications. To visualize metabolite distributions, a free and customizable application, Metabolite Imager, was developed providing several tools for spatially-based chemical data analysis. These tools collectively enable new forms of visualizing the plant lipidome and should prove valuable toward addressing additional unanswered biological questions.

Adipose tissue is a type of connective tissue whose purpose was once thought to be limited to fat storage but is now understood to be a key factor in the pathogenesis of different metabolic diseases, including obesity and type-II diabetes. Adipose tissue consists largely of adipocytes, cells responsible for fat and releasing energy in form of lipids. Different classes of fatty acids, such as saturated and unsaturated have different biological effects on adipocytes. Lipid droplets are the primary organelles in adipocytes that store these fatty acids in form of lipids, and the development of engineered adipose tissues would benefit from improved techniques for analysis of lipid droplet composition, distributions, and dynamics based as a function of fatty acid saturation. Conventional microscopic techniques, such as fluorescence microscopy, provides excellent selectivity of lipid-based structures inside adipose tissue cellular structures based on staining with compound dyes. However, fluorescence staining limits multiplex imaging, and requires time consuming steps in preparing the samples for imaging. Therefore, developing a label-free, high resolution imaging platform with sensitivity to lipid composition could enable analysis of structural and compositional differentiation of lipid droplets within adipocytes during differentiation could give valuable insights into the importance of lipid droplets role in metabolism. As an important step towards achieving this goal, a femtosecond based CARS microscopy imaging platform has been developed to perform in vitro, label-free, imaging of fatty acid composition within engineered adipose tissues. / Bioengineering

As fosfolipases A2 secretadas (sFLA2) de veneno de serpente apresentam homologia estrutural e funcional com as sFLA2s do GIIA de mamíferos, cujos níveis estão elevados em doenças inflamatórias, como a aterosclerose. Nesta doença, as células de músculo liso vascular (CMLVs) acumulam corpúsculos lipídicos (CLs) e se diferenciam em células espumosas. Porém, o papel das sFLA2s neste fenômeno não é conhecido. Neste estudo foram avaliados os efeitos das FLA2 MT-III, cataliticamente ativa, e da BthTx-II, sem atividade catalítica, em CMLVs, com ênfase na formação de CLs e a participação de fatores da homeostasia lipídica. Os resultados obtidos demonstraram que a MT-III e a BthTx-II induziram a formação de CMLVs espumosas. Para tanto, estas enzimas recrutaram diferentes fatores envolvidos na síntese e acúmulo de lipídios. Nesta condição, os CLs constituem um local de síntese de prostaglandinas. Ainda, a MT-III induziu a diferenciação de CMLVs para fenótipo e função de macrófagos. A atividade catalítica não é relevante para a formação de CLs induzida por FLA2s. / Bothrops snake venom secreted phospholipases A2 (sPLA2s) share structural and functional features with mammalian GIIA sPLA2s, which are highly expressed during inflammatory diseases, such as atherosclerosis. In this disease, vascular smooth muscle cells (VSMCs) are loaded with lipid droplets (LDs) differentiating into foam cells. However, the role of these enzymes in this process is still unknown. In this study the effects of snake venom PLA2s MT-III with catalytic activity and BthTx-II, devoid of catalytic activity in VSMCs, with focus on LDs formation and mechanisms involved were investigated. Results here obtained show that both MT-III and BthTx-II induce formation of foam VSMCs and recruit distinct factors of synthesis and storage of lipids in these cells. In this condition, LDs constitute sites for synthesis of prostaglandins. Moreover, MT-III showed the ability to modulate VSMCs functions, leading them to a phenotipic switch to macrophage-like cells. In addition, the catalytic activity is not relevant to sPLA2-induced LDs formation.