1,721,317 research outputs found
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Mechanisms that Contribute to Retinal Pigment Epithelium Dysfunction in Macular Degenerations
No full textMacular degenerations impact millions across the globe, with this number predicted to grow due to lack of effective therapies to combat the disease. The complex etiology of macular degenerations makes their developing treatments difficult. This thesis seeks to explore the underlying mechanisms and how they contribute to the disease in three ways. First, I show how ceramide accumulation drives the release of extracellular vesicles from the retinal pigment epithelium. These extracellular vesicles contain pro-inflammatory cargo and are predominantly released from the apical surface of the retinal pigment epithelium, towards the retina. This disrupts communication in the retina and recruits harmful microglia into the subretinal space. Second, I show that the retinal pigment epithelium are uniquely mechanosensitive to their environment, such that “drusen-in-a-dish” models, where I use biologically inert silica beads, can drive mitochondrial fragmentation in retinal pigment epithelium and disrupt retinal pigment epithelium homeostasis. These can be extrapolated towards mechanisms of disease in age-related macular degeneration. Finally, I show that an Food and Drug Administration-approved drug for osteoporosis and Paget disease, zoledronic acid, can be repurposed to reduce ceramide levels within the retinal pigment epithelium. And by reducing ceramide levels, many of the disruptions to retinal pigment epithelium function can be ameliorated and improve vision in mouse models of inherited macular degenerations. Together, these findings can be used for a better understanding of macular degenerations and hopefully pave the way for a curative intervention
Developing Small-Molecule Regulators of the Hsp70/Hsp40 Complex
No full text24 p.Alzheimer’s Disease is a tauopathy, meaning that misfolded tau proteins
aggregate in the extracellular matrix causing gradual neurodegeneration. Research has
shown the chaperone protein Hsp 70 to be a key factor in the formation of protein
aggregates that contribute to the pathology of the disease. Hsp 70 works in concert with
Hsp 40 to regulate protein folding, refolding and degradation. Inhibition of Hsp 70 has
been shown to decrease protein aggregates. Flavanoids such as myricetin have proven to
be effective inhibitors of Hsp70 but very poor drug candidates. A molecular modeling
program was used to create a list of myricetin-analogs that were then tested for inhibitory
potential by the Malachite Green Assay, Quinaldine Red Assay and ELISA. All thirteen
chosen analogs proved to be ineffective inhibitors of the Hsp 70/Hsp 40 complex, but
provided valuable insight into the interaction between myricetin and Hsp 70. The
previously proposed myricetin binding site was likely incorrect and further investigation
into the inhibition mechanism is necessary.Life Science Institute. University of Michigan. Ann Arbor, Michigan
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Large-scale mutational analysis of transporters in the Solute Carrier Family 22: applications in rare disease and pharmacogenetics
Get PDFFrom disease diagnostics to precision dosing of medication, genome sequencing has the potential to revolutionize healthcare. A key challenge in translating genetic information into clinical action is understanding the phenotypic consequences of genetic variants in clinically important genes. Transporters encoded by genes in the Solute Carrier (SLC) family 22 have strong clinical relevance in rare genetic disease and pharmacogenetics. For example, loss-of-function variants in SLC22A5, encoding the carnitine transporter OCTN2, cause the rare metabolic disorder Carnitine Transporter Deficiency (CTD), and variants with functional consequences in SLC22A1, encoding the hepatic uptake transporter OCT1, contribute to interindividual differences in exposure and response for many commonly used medications. Experimental studies to uncover phenotypic consequences of coding region variants in transporters and other genes have struggled to match pace with the rate at which variants are identified by next-generation sequencing, slowing translation into clinically actionable information. The goal of this dissertation research is to experimentally and computationally address the key challenge of understanding the phenotypic effects of genetic variants in SLC22 transporters, with a primary focus on OCTN2 and OCT1. The dissertation begins with an overview of current practices and limitations in the interpretation of variation in genes underlying inborn errors of metabolism and drug response, detailing experimental approaches to validating a variant as causative or pathogenic and summarizing advances in computational methods aiming to predict variant effect on protein function. We propose a vision for a genomic learning healthcare system (GLHS) that facilitates the translation of a patient’s genome into clinically actionable information for diagnostic and therapeutic purposes. After the overview, we present a rich set of experimental and computational approaches, which were developed and used to improve the functional prediction of genetic variants in OCTN2 for diagnosis of CTD. We functionally characterized 150 OCTN2 missense variants and found that 71% of variants had a significant effect on the uptake of carnitine. 25% of variants reduced transporter function to less than 20% of the wild-type OCTN2, a clinically meaningful threshold for CTD. We asked what was causing reduced function, and identified improper subcellular localization to be a major loss-of-function mechanism affecting 62% of variants. These data were then used in machine learning to build a protein-specific variant effect prediction model that accurately classified variants of OCTN2 as functional (>20%) or LOF (<20%) (area under the receiver operating characteristics curve 0.895±0.025). The machine learning models outperformed current models in terms of functional predictions of genetic variants in OCTN2. Limitations, however, included the number of variants experimentally tested to inform the models, which were limited by experimental methodologies. Therefore, we asked how we can increase throughput of SLC transporter variant phenotyping and transfer predictive models to other SLC22 family members. To this end, we developed a platform for deep mutational scanning (DMS) of a homolog of OCTN2 in the SLC22 family, OCT1 (SLC22A1) to increase the scale and diversify the phenotypes with which we can investigate functional genomics of transporters. We generated a landing-pad based cell system for expression of OCT1 and validated the system with multiple assays involving diverse substrates and phenotypes: uptake of the fluorescent substrate ASP+; uptake of the radiolabeled substrates MPP+ and metformin, and cytotoxicity of the OCT1 substrates, oxaliplatin and platinum analogs SM73 and SM85. We confirmed that OCT1 variants exhibit substrate-specific functional effects with variants p.R61C, p.P117L, and p.G401S. Then, we constructed a library of all 11,572 possible missense and single amino acid deletion variants to undergo functional and spatial characterization by the established DMS system. Data generated with this platform will be useful in the interpretation of OCT1 variants and clinically actionable for drug dosing purposes in precision medicine. In summary, this dissertation research led to a top performing model for predicting the functional effects of variants in OCTN2, which may be causal for CTD. Importantly, we addressed limitations in our model and developed experimental methodologies that extended both the scale of the genetic variants under investigation and the functional phenotypes assessed. Collectively, these methods together with artificial intelligence including machine and transfer learning methods should lead to comprehensive models for accurately predicting the function of coding region variants of all genes in the SLC22 family. These studies will pave the way to a new understanding of the effects of genetic variants in SLC transporters that are causal for human disease and diverse pharmacogenetic phenotypes
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The Chemical Features of Polyanions Modulate Tau Aggregation and Conformational States
Get PDFThe aggregation of tau into insoluble fibrils is a defining feature of neurodegenerative tauopathies. However,tau has a positive overall charge and is highly soluble; so polyanions, such as heparin, are typically required
to promote its aggregation in vitro. There are dozens of polyanions in living systems and it is not clear which
ones might promote this process. Here, we systematically measure the ability of 37 diverse, anionic
biomolecules to initiate tau aggregation, using either wild type (WT) tau or the disease associated P301S
mutant. We find that polyanions from many different structural classes can promote fibril formation and that
P301S tau is sensitive to a greater number of polyanions (28/37) than WT tau (21/37). We also find that
some polyanions preferentially reduce the lag time of the aggregation reactions, while others enhance the
elongation rate, suggesting that they act on partially distinct steps. From the resulting structure-activity
relationships, the valency of the polyanion seems to be an important chemical feature, such that anions
with low valency tend to be weaker aggregation inducers, even at the same overall charge. Finally, the
identity of the polyanion influences fibril morphology, based on electron microscopy and limited proteolysis.
These results provide insight into the crucial role of polyanion—tau interactions in modulating tau
conformational dynamics with implications for understanding the tau aggregation landscape in a complex
cellular environment
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Chemical and genetic perturbations reveal wiring of the protein homeostasis network in prostate cancer
Get PDFThe protein homeostasis (proteostasis) network is composed of multiple components and pathways that work together to govern protein production, folding, stability and degradation. Cancer cells are especially dependent on the proteostasis network, due to their high protein load, genomic instability, and oncogenic signaling pathways. For this reason, inhibitors of the proteostasis network have been developed as anti-cancer therapies, with varying success. In this dissertation, I will discuss two adjacent strategies we employed to better understand wiring of the proteostasis network in cancer cells. In this first approach, we used existing chemical inhibitors of various proteostasis targets, such as molecular chaperones and the proteasome, and tested them in combination to reveal patterns of synergy and antagonism. In the second approach, we used a functional genomics shRNA screening approach to probe at genetic vulnerabilities in the proteostasis network and identify new targets. In both these studies, we focused our work on prostate cancer, due to it’s established dependence on molecular chaperones. Through these approaches we were able to identify both synergistic drug combinations and new proteostasis targets that can be further explored as anti-cancer therapeutic strategies. Furthermore, we gained a better understanding of proteostasis network wiring in prostate cancer cells, which provides broader insights into how to can be therapeutically targeted in cancer
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Discovery and characterization of chaperone-independent substrates of the CHIP ubiquitin ligase
Get PDFProtein turnover through endolysosomal degradation and the ubiquitin proteasome system are critical for maintaining protein homeostasis. These pathways protect the cell from accumulation of misfolded proteins, coordinate critical signaling processes, and facilitate recycling pathways that are central to cellular health. An integral member of these systems is the E3 ubiquitin ligase CHIP, which facilitates the turnover of damaged or terminally misfolded proteins. Canonically, substrate recognition by CHIP is dependent on Hsp70 or Hsp90 chaperones, which serve as intermediaries between a misfolded client and CHIP. However, emerging evidence suggests that CHIP also has the capacity to recognize substrates independent of a chaperone binding partner. In this dissertation, we explore the biological relevance of such chaperone-independent substrates by exploring interactions predicted by a biophysical scoring function we previously developed, termed CHIPscore.In the first chapter, I and others describe one such interaction between CHIP and the relatively uncharacterized, membrane-anchored protein CHIC2. We find that CHIC2 binding strongly attenuates CHIP activity, and that CHIC2 knockout phenocopies CHIP knockout in certain cell types, implying that chaperone-independent interactions can sometimes predominate CHIP’s biological functions. Furthermore, loss of the CHIP-CHIC2 interaction induces neurodegeneration and shortens lifespan in C. elegans, demonstrating that formation of this chaperone-independent complex is important in animals. We propose that CHIC2 attenuates CHIP activity at the membrane, offering a novel mechanism by which this ubiquitin ligase can be regulated.In the second chapter, I explore additional chaperone-independent interactors beyond CHIC2 that are predicted by CHIPscore. In preliminary results, I demonstrate interactions between CHIP and three additional proteins. These proteins are completely uncharacterized and reside at different subcellular localizations, suggesting that CHIP may be regulated at various locations within the cell through mechanisms that are not yet understood. This work opens substantial new avenues upon which future studies should be based
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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Investigation of the physical and genetic interactions between molecular chaperones and their clients
Get PDFMolecular chaperones bind to misfolded proteins (“clients”) to protect them from aggregation and promote their folding. The following Chapters all focus on understanding how the protein-protein interactions (PPIs) between members of the molecular chaperone network and their client proteins regulate these processes. Specifically, I develop a functional genomic approach to explore which molecular chaperones are required for the stability of specific client proteins (Chapter 2) and use biophysical and biochemical approaches to probe how conserved amino acid sequences in the heat shock protein 70 (Hsp70) chaperone allow it to engage with two different classes of co-chaperones (Chapter 3). Together, these studies and recent literature reports suggest that weak interactions (i.e. micromolar) are critical to the molecular logic of chaperone-mediated folding
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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