Browsing by Subject "Serine Endopeptidases"
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Item ApoE Receptors in Alzheimer's and CNS Function(2017-03-24) Lane-Donovan, Courtney; Powell, Craig M.; Herz, Joachim; Eisch, Amelia J.; Huber, Kimberly M.Alzheimer's disease (AD) is a currently incurable neurodegenerative disorder and the most common form of dementia over age 65. The predominant genetic risk factor for AD is the ε4 allele of apolipoprotein E (ApoE4). Other genes related to lipid metabolism and lipoprotein receptor signaling have also been identified as risk modifiers for AD. Despite nearly two decades of research, the mechanisms by which lipid transport proteins cause central nervous system (CNS) disease are not completely understood. Here, the ApoE receptor family and its ligands and their roles in CNS function and neurological disease were explored. It has been previously shown that ApoE4 renders neurons resistant to the neuromodulator and ApoE receptor ligand Reelin, which enhances synaptic plasticity and protects against amyloid β (Aβ) oligomer-induced synaptic toxicity in vitro. Here, mice with reduced Reelin expression were more sensitive to amyloid-induced synaptic suppression, and had memory and learning disabilities at very low amyloid levels. However, this effect of Reelin loss did not extend to other forms a neurological insult, since the Reelin conditional knockout mice were not more susceptible to transient middle cerebral artery occlusions. Together, these findings highlight the specific physiological importance of Reelin for protecting the brain against Aβ-induced synaptic dysfunction and memory impairment. One of the continuing debates in the AD field is whether ApoE is required for synaptic function. ApoE knockout mice have synaptic loss; however, they also have severely increased plasma lipids, which could independently contribute to CNS dysfunction. A novel mouse with normal plasma ApoE, but severely depleted brain ApoE, shares a similar synaptic phenotype as ApoE knockout mice, suggesting central ApoE is required for brain function. To determine if diet can modulate ApoE levels, wildtype, ApoE3, and ApoE4 targeted replacement mice were fed a chow, high-fat, or ketogenic (high-fat, very-low-carb) diet. Surprisingly, high-fat diet reduced hippocampal ApoE levels in ApoE3 TR mice, indicating an intersection of genetic (ApoE isoform) and lifestyle (diet) risk factors on AD pathogenesis. Taken together, these findings highlight the importance of ApoE receptors and their ligands in AD biology, and future studies will have to determine how to target these mechanisms to treat AD and improve patient outcomes.Item Characterization of the Non-Proteolytic Mechanism and Cellular Site of Action of PCSK9-Mediated Degradation of the Low-Density Lipoprotein Receptor(2010-05-14) McNutt, Markey Carden, II; Horton, Jay D.Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is a serine protease that has emerged as a central regulator of plasma low-density lipoprotein cholesterol levels. Here, it is demonstrated that PCSK9 is secreted into the blood and that the secreted PCSK9 binds and degrades LDLRs in liver. To determine if PCSK9 catalytic activity was involved in the degradation of the LDLR, a mutant PCSK9 was engineered that is catalytically inactive. Studies with catalytically inactive PCSK9 demonstrated that the protein degraded LDLRs in a manner that was indistinguishable from wild-type PCSK9, suggesting that proteolytic activity is not required for PCSK9-mediated degradation of the LDLR. Crystallographic analysis of a PCSK9:LDLR complex supported the experimental findings that PCSK9 does not catalytically cleave LDLRs. These studies also suggested that one therapeutic approach for treating hypercholesterolemia might be to disrupt the PCSK9:LDLR interaction at the cell surface. Recombinant LDLR subfragments were synthesized and added to the medium of cells that overexpressed PCSK9. These sub-fragments restored LDLRs to levels found in the control cells. These experiments confirmed that the disruption of PCSK9:LDLR at the cell surface can inhibit PCSK9 activity and suggested that the majority of PCSK9 activity is extracellular. Further structural analysis of the PCSK9:LDLR co-crystal predicted that an LDLR mutation (His306Tyr) might increase the affinity of PCSK9 for the LDLR, and thus could be associated with familial hypercholesterolemia (FH). A search of the LDLR mutation database revealed that the LDLR mutation (His306Tyr) had been reported in a kindred with FH. Structure/function studies with the mutant LDLR(H306Y) protein showed that this mutation mimics a conformation change in the wild-type LDLR that occurs at low pH, which results in increased binding. The increased affinity between PCSK9 and LDLR(H306Y) promoted enhanced LDLR degradation. Thus, this mutation represents a previously unrecognized class (Class VI) of FH mutants. Finally, two high-throughput assays were developed to discover new small molecule inhibitors of intracellular PCSK9 autocleavage and to identify previously unrecognized protein activators of PCSK9 action. Use of these assays could provide additional avenues for modulating PCSK9 activity and lead to new therapeutic options for the treatment of hypercholesterolemia.Item Does Reelin Affect Recovery from a Stroke?(2016-01-19) DeSai, Charisma; Herz, Joachium; Stowe Ann M.; Lane-Donovan, CourtneyReelin is an extraceullular glycoprotein that modulates synaptic plasticity and increases long-term potentiation. Since Reelin has neuroprotective effects, we were interested to see if Reelin plays a role in recovery after significant neurological damage. Earlier studies with reeler mice showed that mice lacking Reelin have increased susceptibility to stroke and suffer more damage post-stroke. Since Reelin is important in neuronal migration during development, it is possible that the effects seen were due to improper brain development, instead of Reelin deficiency itself. Instead of using Reeler mice, we used the Reelin conditional knockout(cKO) mouse model. Thus, we were able to see the effects of Reelin loss while permitting normal brain development. Stroke size and post-stroke behavior were investigated after inducing transient middle cerebral artery occlusion in four-month-old Reelin cKO mice. No significant difference was seen between wild type and Reelin cKO mice in infarct size or behavior, suggesting that while Reelin does play in important function in the brain, it does not play a significant role in post-stroke recovery.Item Transmembrane Protease TMPRSS11B Promotes Lung Cancer Growth by Enhancing Lactate Export and Glycolytic Metabolism(2018-11-26) Updegraff, Barrett Logan; Buszczak, Michael; O'Donnell, Kathryn A.; Mendell, Joshua T.; Morrison, Sean J.Pathways underlying metabolic reprogramming in cancer remain incompletely understood. We identified the Transmembrane Serine Protease TMPRSS11B as a novel gene that promotes transformation of immortalized human bronchial epithelial cells. TMPRSS11B is upregulated in human lung squamous cell cancers and high expression is associated with poor survival of non-small cell lung cancer patients. TMPRSS11B depletion in human lung squamous cell cancer reduced transformation and tumor growth. TMPRSS11B harbors an extracellular protease domain and we hypothesized that catalysis of a membrane bound substrate modulates tumor progression. Interrogation of a set of soluble receptors revealed that wild-type, but not catalytic mutants of TMPRSS11B promotes membrane release of Basigin, an obligate chaperone of the lactate monocarboxylate transporters MCT1 and MCT4. To investigate whether TMPRSS11B regulates lactate transport, we monitored intracellular lactate content and lactate secretion. Our data suggest TMPRSS11B regulates cellular lactate levels by interacting and co-localizing with Basigin and MCT4 at the plasma membrane to enhance their lactate export efficiency. Specifically, TMPRSS11B expression promoted lactate secretion concomitant with reduced levels of intracellular lactate content. Conversely, TMPRSS11B depletion in lung squamous cell cancer lines resulted in substantial accumulation of intracellular lactate. We detail a novel metabolic role of TMPRSS11B and this work identifies an oncogenic transmembrane protease that promotes tumorigenesis, thereby uncovering a new enzymatic activity that may be targeted for cancer therapy. Interrogation of lactate metabolism in vivo will ultimately guide these therapies and contribute to our growing knowledge of lactate biology.