Regulation of Diacylglycerol Acyltransferase-2 and Triacylglycerol Synthesis by Protein Ubiquitination

Triacylglycerol is the major source of stored energy in humans, yet, excessive accumulation of triacylglycerols in tissues leads to obesity, diabetes and heart disease. Triacylglycerol synthesis is catalyzed by diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Although recent studies have shed light on the metabolic functions of these enzymes, little is known about their regulation. We have found that DGAT2 is a short-lived protein and is degraded via the ubiquitin- proteasome pathway. Our objective was to identify the lysine residues that are ubiquitinated and determine the role of ubiquitination in regulating DGAT2 stability and triacylglycerol synthesis. Initial experiments found that a lysine-less DGAT2 mutant (Lys-less-DGAT2) was not degraded. Moreover, Lys-less-DGAT2 exhibited altered subcellular localization and disrupted lipid droplet biogenesis. Screening of a DGAT2 lysine-to-arginine mutant library demonstrated that several lysine residues are involved in regulating DGAT2 stability. Substitution of two lysine clusters was sufficient to mislocalize DGAT2 and perturb typical lipid droplet formation, suggesting that these lysines may have a role in targeting DGAT2 to lipid droplets. Interestingly, DGAT2 on lipid droplets was ubiquitinated and stimulating lipogenesis did not reduce DGAT2 degradation. Monoacylglycerol acyltransferase (MGAT) enzymes, MGAT2 and MGAT3, are closely related to DGA T2 and produce the DGA T2 substrate, diacylglycerol. MGA T2 and MGA T3 interact with DGAT2 and appear to stabilize it. We determined that DGAT2, MGAT2 and MGAT3 are targeted for endoplasmic-reticulum-associated degradation (ERAD), as ERAD inhibition caused poly-ubiquitinated species of all three proteins to accumulate. Moreover, overexpression of DGAT2 and MGAT2 resulted in redistribution of the ERAD ATPase, valosin-containing protein (VCP/p97), where it becomes concentrated in the ER, co-localizing with both DGAT2 and MGAT2. Interaction of DGAT2 and MGAT2 with VCP/p97 was also demonstrated in situ. We took a non-targeted mass spectrometry approach to identify proteins interacting with DGAT2. The carbohydrate binding protein, calnexin, was one of the candidates identified. This interaction was confirmed in vitro and in situ. The possible impacts of calnexin on triacylglycerol metabolism were examined in calnexin knockout mouse embryonic fibroblasts, which exhibited stunted lipid droplet size compared to wild-type cells. Collectively, these investigations provide insight into the post-translational regulatory mechanisms of DGAT2 and DGAT2 family members

An Absolutely Conserved Motif of the Essential Translocon Subunit, Sss1, Dictates Protein Function and Stability with Implications for Personalised Medicine

ER channels and components in translocation are frequently described with roles in disease progression. We have characterised a highly conserved region of the essential translocon subunit, Sss1, which encodes the proteins degron and contributes to regulating translocon dynamics. This work has utilised these findings to design a yeast-based system to identify novel regulators of translocon dynamics, assess the impact translocon dynamics has in disease and may have future applications as part of drug discovery

Master of Science

thesisChediak-Higashi Syndrome (CHS) is a rare, autosomal recessive genetic disorder caused by mutations in the lysosomal trafficking regulator gene (CHS1) that affects vesicle morphology. The CHS1 gene encodes a protein, Lyst, which belongs to a family of proteins containing a conserved BEACH (BEige And Chediak-Higashi) domain and WD40-repeat domain. The beigej mouse has been the best-studied animal model for CHS. Factor associated with neutral sphingomyelinase activation or FAN is the smallest member of the BEACH family. FAN interacts with the Tumor Necrosis Factor Receptor (TNFR1) to activate Neutral SphingoMyelinase2 (NSMase2). The absence of Lyst or FAN gives rise to enlarged lysosomes. Loss of both proteins results in an additive effect, as demonstrated by increased lysosome size in FAN-/- /beigej mouse. An additive phenotype suggests that there are at least two pathways to regulate lysosome size. Overexpression of Lyst or FAN results in smaller than normal lysosomes. Overexpression of FAN in the absence of Lyst does not give rise to small lysosomes. NSMases are present on lysosomes and inhibition of NSMases, using GW4869 or 3-OMethylsphingomyelin, results in enlarged lysosomes. Further, the decrease in lysosome size seen in FAN-overexpressing cells is blocked by GW4869. These results suggest that FAN activation of NSMase at the lysosome is a crucial step in regulating lysosome size

Intracellular delivery by membrane disruption: Mechanisms, strategies, and concepts

© 2018 American Chemical Society. Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo typesñYsmall molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications and challenges to implementation. Throughout, we highlight specific mechanisms of membrane disruption and suggest areas in need of further experimentation. We hope the concepts discussed in our review inspire scientists and engineers with further ideas to improve intracellular delivery

Antidepressant-like Effects of Peripheral Reelin Administration in a Preclinical Model of Depression

Depression is a serious psychiatric disorder characterized by a range of debilitating symptoms. Long-term exposure to stress is a significant risk factor for the onset and maintenance of depression. Rats exposed to repeated treatment with the stress hormone corticosterone (CORT), a well-established rodent model of depression, begin to display depression-like symptoms. The development of depression-like symptoms with prolonged exposure to CORT is accompanied by reductions in the number and maturation rate of immature dentate granule cells within the hippocampus. Furthermore, these changes are paralleled by gradual decreases in reelin-expressing cells in the dentate subgranular zone of the hippocampus. Reelin is a large extracellular matrix protein that has been implicated in a number of neuropsychiatric disorders, including szchiphrenia, bipolar disorder, autism, as well as major depression. It holds important roles in learning and memory, cell migration and integration, synaptic contact formation, and adult neurogenesis. Mice deficient in reelin are more susceptible to CORT-induced impairments in hippocampal neurogenesis and the development of a depressive phenotype. Previous work has shown that intra-hippocampal infusions of reelin into the hippocampus reverse CORT-induced increases in depression-like behavior in rats, while restoring accompanying impairments in hippocampal neurogenesis. Reelin is also expressed in peripheral organs and tissues, though its roles here are not well understood. However, reelin-deficient mice show peripheral alterations in the clustering pattern of the serotonin transporter (SERT) in membranes of blood lymphocytes. The serotonin transporter is one of the main targets of antidepressant action, and importantly, this altered pattern of SERT clustering in reelin-deficient mice is mirrored both in patients with depression and in rats exposed to prolonged CORT treatment. Based on the previous findings, we were motivated to examine whether peripheral injections of reelin could restore CORT-induced increases in depression-like behavior. To investigate possible mechanisms, we examined (i) the SERT clustering pattern in peripheral lymphocyte membranes, and (ii) the maturation rate of immature hippocampal neurons. 40mg/kg of CORT was administered subcutaneously once per day for 21 consecutive days. In conjunction, we utilized a novel reelin injection paradigm, where reelin was delivered via the lateral tail vein at either 3μg/ml or 5μg/ml every 5 or 10 days during the period of CORT injections. Depression-like behavior was measured using the forced swim test the day following the last CORT injection. The open field test was included as a measure of locomotive and anxiety-like behavior. Importantly, peripheral reelin at all dosages administered restored CORT-induced increases in depression-like behavior in the forced swim test, normalizing both immobility and swimming behaviors. Neither CORT nor reelin impacted open field behavior. As expected, CORT-treated rats displayed alterations in SERT membrane protein clustering, and importantly this was restored by all doses of reelin administered. Peripheral reelin did not significantly reverse the CORT-induced deficits in immature neuron maturation rate. These novel findings demonstrate that reelin has antidepressant-like actions when given peripherally and provide evidence for the regulation of serotonin transporter clustering in lymphocyte membranes as a mechanism for the antidepressant action of reelin

Modulating Hallmarks of Cholangiocarcinoma

How are cholangiocarcinoma cells different from non-malignant cholangiocytes? All of the hallmarks of cancer apply- those reported in this dissertation include cell proliferation, migration, apoptosis, and evasion of growth suppression. My studies began with testing how apoptosis might be regulated through embelin, a small molecule reported to sensitize cells to apoptosis by blocking XIAP. My data however revealed that embelin reduced the proliferative capacity in cholangiocarcinoma cells, but did not increase cell death. Malignant cells exhibit dysregulation of microRNA processing and expression. Hence, my studies seeking ways that cholangiocarcinoma eludes apoptosis transitioned to the oncomiR miR-106b, which is overexpressed in cholangiocarcinoma. I observed that miR-106b protected cholangiocarcinoma cells from apoptosis. Genome-wide screening identified the landscape of miR-106b-targeted genes in a cholangiocarcinoma cell line, some with roles in tumor biology. MiR-106b targets included members of the Krüppel-like factor (KLF) family of transcription factors. The function of KLF2 in biliary epithelia or cholangiocarcinoma is unknown. I describe in part how the cholangiocyte senses the environment through the primary cilium and translates this to regulation of KLF2, a flow-responsive regulatory protein and tumor suppressor in several cancers. I observed lower expression of KLF2 in malignant cholangiocarcinoma cells and tumors compared to normal, and its enforced expression inhibited proliferation and migration while reducing sensitivity to apoptosis. In the normal bile duct epithelium, environmental cues are detected by the cholangiocyte primary cilium, a sensory organelle that functions as a signaling nexus for the cell. Cholangiocarcinoma cells are highly proliferative despite extracellular signals to remain quiescent. Cholangiocarcinoma cells often lose their cilia, resulting in altered communication and unchecked cell growth. I identified a cholangiocyte signaling axis in which the primary cilium maintains quiescence through enhanced KLF2 expression. I present the first finding of a ciliary-dependent KLF2 flow response in cholangiocytes. Overall, this dissertation sought deeper understanding of biochemical and molecular features of cholangiocarcinoma and added to our understanding of microRNAs and mechanosensory pathways

Engineered scaffolds for the preservation of gliding tissue interfaces

The production of modular scaffolds that allow for the facile spatial localisation of a variety of interchangeable functional groups gives a toolbox of options for tissue engineering. Furthermore, the need to re-engineer a system for each specific application or function is eliminated. A modular scaffold system based on the versatility of the electrospinning processing technique, chemical modification of bioresorbable poly-ε-caprolactone (PCL) and a controlled free radical polymerization technique is proposed. An antifouling surface is prepared by grafting a poly(oligoethylene glycol) bottlebrush (pOEGMA) from a surface initiating group on silicon wafers and electrospun PCL scaffolds. The surface initiated polymerization method provides versatility which allows for wide variety of monomer units to be selected for bespoke scaffolds and tailored to particular applications, including fluorescent labelling. The antifouling ability of the pOEGMA functionalization is explored and characterised with protein and cell assays. The chemical and mechanical stability of the pOEGMA functionalized scaffolds is explored in a simulated physiological environment over 10 weeks. Following which the chemical and material properties are re-characterised and found to be maintained. A dual functional bilayer scaffold with an antifouling, non-cell adhesive surface and an opposing cell-adhesive surface is then produced. The pOEGMA coating provides the antifouling surface whilst the cell adhesive side is prepared by end-functionalizing the PCL with the cyclic cell binding Arg-Gly-Asp-Ser (RGDS) peptide. Spatial control of the functionality through the mat is achieved by sequential electrospinning of the functionalized polymers. The opposing properties of the surfaces are demonstrated through cell culture and florescent labelling to illustrate clear spatial segregation. Gliding surfaces such as those found in the musculoskeletal systems, the tendon for example, are vulnerable to tethering from scarring following surgical and traumatic injury. This scaffold could offer protection from these scars through the control of cellular migration and may lead to improved patient outcomes.Open Acces

2015 Annual Research Symposium Abstract Book

2015 annual volume of abstracts for science research projects conducted by students at Trinity Colleg

Lentiviral vectors for treatment of haemophilia

Haemophilia A and B are X‐linked recessive disorders caused by defects in coagulation factors (F) VIII and IX, respectively. Severe cases of haemophilia are characterised by episodes of spontaneous bleeding, predominantly into the joints and muscles, and can result in permanent disability and even mortality if left untreated. The haemophilias are compelling candidates for treatment with gene therapy as therapeutic benefit only requires a modest increase in the endogenous coagulation factor level, response to treatment can be easily monitored, and factor expression can be mediated by many cell types in vivo. Integration deficient lentiviral vectors (IDLVs) offer marked advantages over currently used integrating lentiviral vectors (ILVs) as side effects caused by insertional mutagenesis are potentially minimised. Previous work has shown that efficient and sustained transgene expression in non‐dividing cells, such as brain and muscle tissue, using IDLVs can be achieved. ILVs have previously been used to mediate long term expression of coagulation factors in vivo. In this study, we investigated the use of IDLVs as treatment for haemophilia with muscle and liver tissue (primarily nondividing hepatocytes) as principal targets. Transduction efficiency and relative transgene expression in vivo from ILVs and IDLVs were assessed in both tissues, and a number of strategies, including pseudotyping and tissue specific promoters, were utilised to improve targeted expression. Overall, despite achieving sustained transgene expression from IDLVs, in comparison to ILVs, the levels obtained were significantly lower and IDLVs were unable to mediate expression of human FIX at therapeutic levels in liver. Finally, the expression of bioengineered forms of human factor VIII (hFVIII) was assessed using ILVs in vivo after neonatal delivery. Long‐term expression was achieved and a 20‐fold increase in expression was observed after codon optimisation of the hFVIII cDNA sequence. In conclusion, IDLVs can mediate sustained transgene expression in vivo, however, vectors may need to be further optimised for increased expression to achieve clinical benefit for haemophilia patients