R-3 Repository :: Browsing by Author "Segatori, Laura"

Browsing by Author "Segatori, Laura"

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Anticipation of Nitric Oxide Stress in the Human Commensal Fungus Candida albicans
(2013-07-24) Lynn, Jed; Bennett, George N.; Braam, Janet; Segatori, Laura; Stewart, Charles R.
Candida albicans is the most common human commensal fungus, able to colonize host niches such as skin, mouth and gastrointestinal tract. Colonization of diverse microenvironments requires the ability to evade or overcome innate host protection and adapt to rapid transitions between environments with different stresses and nutrient availability. Colonization of the gastrointestinal tract requires passage through the stomach containing toxic levels of nitric oxide, generated from acidification of nitrite in the low pH of the stomach. Although resistance of C. albicans to nitric oxide is mediated by the flavohemoglobin Yhb1, little is known about the physiologically relevant ligands that regulate YHB1 expression. Here I propose the hypothesis that nontoxic saliva chemicals induce YHB1 expression and promote resistance to nitric oxide generated in the stomach. Supporting this hypothesis is the observation that two ions actively concentrated in the saliva – nitrate and thiocyanate – induce YHB1 expression. Indeed, whole-genome transcriptional analysis of C. albicans treated with nitrate or thiocyanate produce gene expression profiles nearly identical to cells treated with nitrite or nitric oxide. Pretreatment of C. albicans with either of these two nontoxic compounds increases resistance of the yeast to nitric oxide. I propose that this is an evolved response in which C. albicans anticipates nitric oxide stress generated in the stomach. C. albicans thus upregulates nitric oxide stress response genes in response to saliva signals that precede nitric oxide formation further on in the gut. Only a few examples of anticipatory signaling have so far been identified and it is not known how common this type of regulation is among microbes. Expression of the YHB1 gene in response to nitric oxide is regulated by the transcription factor Cta4. I show that Cta4 binds to the YHB1 promoter in vivo as a homodimer and is necessary, but not sufficient, for nitric oxide, nitrate and thiocyanate induced expression of YHB1. Based on these data I propose a model in which Cta4 transcriptional activation is inhibited under non-inducing conditions by a negative regulator. Understanding the mechanism by which C. albicans senses and responds to nitric oxide, nitrate and thiocyanate remains a question for future research.
Characterization & Application of Immobilized Biomacromolecules using Microcantilever and QCM Sensors
(2014-04-15) Wang, Jinghui; Biswal, Sibani Lisa; Segatori, Laura; Wong, Michael S.; McDevitt, John T.; Suh, Junghae
The structure and function of immobilized biomacromolecules are likely to be altered because of the solid surface. The long-term objective of this thesis is to develop surface-based biosensors for the characterization and application of biomacromolecules at the liquid-solid interface. In this study, two analytical surface-sensitive sensors are utilized: microcantilevers and quartz crystal microbalance with dissipation (QCM-D). Each offers unique information regarding the molecules of interest. In particular, the systems that are covered in this thesis include the detection of target analytes using specific recognition elements and the characterization of supported lipid membranes. This research has led to a better understanding of the effect of solid surfaces on protein structure and function, as well as the ability to engineer biomolecular surfaces with great control. There are two detection systems that were studied: a phage-derived peptide system for the detection of pathogenic bacteria Salmonella and an antibody displacement assay for the detection of an explosive, 2,4,6-trinitrotoluene (TNT). The microcantilever responds to changes in the surface free energy on the sensor surface by monitoring changes in its deflection. The physisorption or chemisorption of molecules to the cantilever surface induces a mismatch in the surface stress, causing the cantilever to bend. The multiplexed measurement is able to quickly determine the binding affinities of various phage-derived peptides, improving the screening efficiency of the peptides derived from phage display libraries for Salmonella detection. The microcantilever-based technique provides a novel biosensor to rapidly and accurately detect pathogens and holds potential to be further developed as a screening method to identify pathogen-specific recognition elements. QCM measures mass changes on the sensor surface by monitoring the frequency change of the crystal. The combination of a competition assay with QCM using an anti-TNT antibody is able to distinguish a TNT molecule among molecules of similar structure at low concentrations, leading a sensitive and selective assay. The reliability of this method was further investigated in more real environments simulated by fertilizer solution and seawater. Furthermore, this method could be also applied in gas phase detection of TNT, as well as the detection of other chemicals, such as environmental pollutants and illegal drugs. In both of these detection assays, a mathematical model was developed to quantify the binding of the target molecules with the molecules of interest. In the second half of the thesis, the microcantilever sensor is applied to characterize supported lipid bilayers (SLBs), an interesting biomacromolecular assembly that holds great importance as a model system for membranes. Through monitoring the cantilever deflection, the formation of the SLB, its temperature induced phase transitions, and its interactions with membrane-active molecules are investigated. With increasing temperature, the lipid acyl chains transition from an ordered state to a disordered state, accompanied by a changes in the surface stress that can be readily detected using microcantilever. The phase transition temperature of SLBs is different from that of a lipid monolayer, indicating that the existence of the solid support aﬀects the monolayer structure. Two amphipathic membrane-active molecules, peptide (PEP1) and a triblock copolymer (Pluoronic), are studied for their associations with SLBs. PEP1’s association with SLBs highly depends on the ratio of peptide over lipid, while the Pluoronic interacts with SLBs as a function of temperature and the length of lipophilic block in the copolymer. Therefore, the microcantilever sensor is capable of measuring the conformational change of surface-bound molecules, as well as characterizing the kinetics of membrane-peptide interactions with great sensitivity.
CLN8 is an endoplasmic reticulum cargo receptor that regulates lysosome biogenesis
(Springer Nature, 2018) di Ronza, Alberto; Bajaj, Lakshya; Sharma, Jaiprakash; Sanagasetti, Deepthi; Lotfi, Parisa; Adamski, Carolyn Joy; Collette, John; Palmieri, Michela; Amawi, Abdallah; Popp, Lauren; Chang, Kevin Tommy; Meschini, Maria Chiara; Leung, Hon-Chiu Eastwood; Segatori, Laura; Simonati, Alessandro; Sifers, Richard Norman; Santorelli, Filippo Maria; Sardiello, Marco; Bioengineering; Biosciences; Chemical and Biomolecular Engineering
Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment1,2,3. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system4,5,6, but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease)7. ER-to-Golgi trafficking of CLN8 requires interaction with the COPII and COPI machineries via specific export and retrieval signals localized in the cytosolic carboxy terminus of CLN8. CLN8 deficiency leads to depletion of soluble enzymes in the lysosome, thus impairing lysosome biogenesis. Binding to lysosomal enzymes requires the second luminal loop of CLN8 and is abolished by some disease-causing mutations within this region. Our data establish an unanticipated example of an ER receptor serving the biogenesis of an organelle and indicate that impaired transport of lysosomal enzymes underlies Batten disease caused by mutations in CLN8.
Development of a gene signal amplifier platform for monitoring the Unfolded Protein Response
(2019-12-18) Origel Marmolejo, Carlos Alberto; Segatori, Laura
Sophisticated mechanisms controlling regulation of gene expression in mammalian cells mediate the translation of instructions stored in the genome into molecular components that define cellular physiology. Gene expression results from coordinated protein-driven processes guided by regulatory mechanisms that rely on protein-protein interactions, protein localization, and chromatin rearrangement. Characterizing gene expression profiles is critical to understand stress-response signaling pathways that rely on regulation of gene expression to integrate information about the nature, duration, and intensity of stress stimuli. Current technologies to monitor gene expression are mainly based on transcriptomic analyses, such as DNA microarrays, RNA-seq, and quantitative RT-PCR, which do not provide temporal resolution of gene expression dynamics. Reporter gene assays based on minimal or synthetic regulatory sequences enable facile detection of gene expression, but often fail to recapitulate the innate chromosomal regulatory mechanisms. To address the need to monitor gene expression from the native chromosomal context and thus encompassing the complexity of mammalian regulatory systems while generating a readily detectable signal output that recapitulates gene expression dynamics, I developed a gene signal amplifier platform that links transcriptional and post-translational regulation of a fluorescent output to the expression of a target gene. Specifically, I generated a HEK293 master cell line containing a genetic circuit for signal amplification and developed derivatives cell lines engineered to link the expression of chromosomal genes to the genetic circuit for signal amplification using CRISPR-Cas9 technology. Iterations of mathematical modeling and experimental tests led to the development of a cell-based reporter platform that can detect changes in gene expression with high sensitivity, dynamic range, and dynamic resolution. To validate this reporter platform, I generated a multiplex reporter system for monitoring markers of the unfolded protein response (UPR), a complex signal transduction pathway that remodels gene expression in response to proteotoxic stress in the endoplasmic reticulum. I implemented the gene reporter platform to profile the temporal pattern of regulation of genes markers of different branches of the UPR which determines the outcome of UPR activation, namely stress attenuation or apoptosis. By recapitulating the transcriptional and translational control mechanisms underlying the expression of a target gene, this platform provides a novel technology for monitoring gene expression with high sensitivity and dynamic resolution.
Differential Autophagic Responses to Nano-Sized Materials
(2018-04-19) Popp, Lauren Bailey; Segatori, Laura
Recent progress in the field of nanotechnology has led to a dramatic increase in the number of engineered nanomaterials currently being produced and introduced in the marketplace, raising concerns regarding the risk of human exposure to these specially designed, highly reactive materials. Engineered nanomaterials exert a diverse range of effects on biological systems, reflecting the heterogeneity and complexity of their properties. The effects of nanomaterials on biological systems, which range from inflammatory response to carcinogenicity and neurodegeneration, originate from the interaction between nanomaterials and cellular components, which also operate at the nanoscale. Cellular clearance mechanisms are typically activated in response to internalization of nano-sized materials. In particular, the cellular response to nanomaterials is mediated by the lysosome-autophagy system, which is the main catabolic pathway in mammalian cells that mediates degradation of a variety of nano-sized materials that are encountered by the cell through the lysosome. The lysosome-autophagy system is thus at the forefront of the cellular response to the uptake of nanomaterials: depending on the nanomaterial’s physicochemical properties, this response can vary dramatically and may culminate in enhancement of cellular clearance or blockage of the autophagic flux and, eventually, cell death. Due to its important role in maintaining cellular homeostasis and survival, defects in the lysosome-autophagy system may have deleterious effects on cells, possibly leading to pathologic conditions. The objective of this study was to characterize the response of the lysosome-autophagy system to different types of nanomaterials, namely genetically encoded adeno-associated virus (AAV) particles, which are of particular interest due to their potential as gene delivery vectors, and engineered titanium dioxide and zinc oxide nanoparticles, which are used in a variety of consumer products. I investigated a comprehensive set of makers of the lysosome-autophagy system with the ultimate goal of elucidating the specific nature of the autophagic response to nanomaterial uptake and whether it leads to biocompatible or bioadverse effects on cell physiology. Analyses of the molecular mechanisms underlying the autophagic response to AAV nanoparticles revealed that uptake of AAV induces activation of autophagy, which, in turn, results in reduction in transgene delivery. Upregulation of autophagy induced by AAV also causes enhanced degradation of potentially toxic autophagic substrates. These results provide important insights for the design of AAV-based gene delivery systems as well as nanotherapeutics for the treatment of diseases characterized by insufficient autophagic clearance. Titanium dioxide nanoparticles of different primary particle diameters were found to induce transcriptional upregulation of the lysosome-autophagy system. Prolonged exposure to titanium dioxide nanoparticles, however, induced lysosomal membrane permeabilization, leading to blockage of autophagic flux and accumulation of autophagic substrates. These results point to the complexity of the autophagic response to nanomaterials, which may involve activation of the lysosome-autophagy system, but also impairment of some of its components, leading to accumulation of autophagic vesicles that cannot be cleared by lysosomes. Exposure to zinc oxide nanoparticles was also found to cause transcriptional upregulation of the lysosome-autophagy system and formation of autophagic vesicles. Cell treatment with bare zinc oxide nanoparticles (~85 nm) or coated with a highly stable silicone derivative (triethoxycaprylylsilane) enhanced the formation and turnover of autophagosomes, while exposure to larger zinc oxide particles (~200-1000 nm) caused blockage of autophagic flux, resulting in accumulation of autophagosomes. Results from this study provide important insights into the effect of nanomaterials on the lysosome-autophagy system and will inform the design of the next generation of nanomaterials with predictable autophagy-modulating properties for a variety of industrial and medical applications.
Encryption of Adeno-Associated Virus for Protease-Controlled Gene Therapy
(2013-09-16) Judd, Justin; Suh, Junghae; Silberg, Jonathan J.; Segatori, Laura
Gene therapy holds the unprecedented potential to treat disease by manipulating the underlying genetic blueprints of phenotypic behavior. Targeting of gene delivery is essential to achieve specificity for the intended tissue, which is especially critical in cancer gene therapy to avoid destruction of healthy tissue. Adeno-associated virus (AAV) is considered the safest viral vector and, compared to non-viral vectors, offers several advantages: higher efficiency, genetic modification, combinatorial panning, and high monodispersity. Classic viral targeting has focused on engineering ligand-receptor interactions, but many cell surface targets do not support post-binding transduction events. Furthermore, many potential target tissues – such as triple negative breast cancer – may not display a single, unique identifying surface receptor, so new methods of targeting are needed. Alternatively, many pathological states, including most cancers, exhibit upregulation of proteolytic enzymes in the extracellular milieu. The present work describes the development of an AAV platform that has been engineered to activate in response to disease-related proteases. The specificity and sensitivity of these protease-activatable viruses (PAVs) can be tuned to meet the demands of various clinical scenarios, giving the platform some therapeutic versatility. This work represents the first demonstration of a protease-controlled, non-enveloped virus for genetic therapy. These results extend the therapeutic value of AAV, the safest gene vector currently being explored in 73 clinical trials worldwide.
Expanding the Mammalian Synthetic Biology Toolbox: Orthogonal Regulators and Gene Circuits for Monitoring Protein Folding and Degradation
(2019-04-11) Zeng, Yimeng; Segatori, Laura
Engineering mammalian cells holds great promise for a variety of biomedical applications, ranging from the design of model systems to study complex biological processes underlying human diseases, to the development of cell-based therapies and the production of therapeutic biomolecules. Cell engineering requires sophisticated molecular tools that can interface with cellular systems, but also provide orthogonal functionalities, to achieve precise control over complex gene networks. In an attempt to expand the mammalian synthetic biology toolbox for applications in the study of protein misfolding diseases, my research seeks to design and construct orthogonal gene regulators and genetic circuits to monitor protein folding and degradation. To develop a cell-based sensor for monitoring protein aggregation, I engineered a split transcriptional repressor that links protein aggregation to expression of an easily detectable reporter. I designed a set of two-fragment tetracycline repressor (TetR) variants that can function as transcriptional AND gates in both bacteria and mammalian cells. I built a protein solubility sensor by co-expressing the large “detector” fragment of the split TetR and a small “sensor” fragment fused to the target protein, and demonstrated that protein aggregation can be detected by monitoring complementation between the “detector” and “sensor” fragments. This split TetR represents a novel genetic component that can be used in bacterial as well as mammalian synthetic biology and a much-needed cell-based sensor for monitoring protein conformation in complex cellular environments. To develop a tunable, hysteretic sensor for detection of proteasomal degradation, I built a genetic circuit (Hys-Deg) based on a self-activation loop consisting of a tetracycline-controlled transactivator (tTA) variant engineered to interface with the ubiquitin proteasome system (UPS). Guided by predictive modeling, I demonstrated that control of the hysteretic response is achieved by modulating the ratio of expression of constitutive to inducible tTA that generates the self-activation loop. I also showed that the system can be finely tuned through dosage of tetracycline to calibrate the circuit for detection of desired levels of UPS activation. This study establishes the design rules for building a hysteretic circuit with an autoregulatory feedback loop and provides a synthetic memory module that can be integrated into regulatory gene networks to study and engineer complex cellular behaviors.
Expanding the mammalian synthetic biology toolbox: Orthogonal tools and circuits for cellular reprogramming
(2020-12-04) Bachhav, Bhagyashree; Segatori, Laura
Cell engineering presents great potential for next-generation biotechnological and biomedical applications that employ engineered mammalian cells to sense intracellular or extracellular signals and respond in a user-defined manner. Programming mammalian cells requires tools that operate orthogonally to the cellular machinery for sensing and controlling the levels, localization, and function of endogenous proteins. This work describes my efforts to expand the mammalian synthetic biology toolbox and develop orthogonal tools and gene circuits to reprogram cells for a diverse range of applications. I first illustrate the development of a gene signal amplifier platform to monitor chromosomal gene expression that recapitulates gene expression dynamics from the native chromosomal context and generates a readily detectable signal output. The gene signal amplifier links transcriptional and post-translational regulation of a fluorescent output to the expression of a chromosomal target gene. By recapitulating the transcriptional and translational control mechanisms underlying the expression of a target gene with high sensitivity, this platform provides a novel technology for monitoring target gene expression with superior sensitivity and dynamic resolution. To illustrate the utility of such a reporter system, I used the gene signal amplifier platform to monitor the activation and dynamics of the unfolded protein response (UPR), a complex signal transduction pathway that remodels gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER). The UPR manifests as a series of signaling cascades aimed at relieving proteotoxic stress and restoring homeostasis or executing apoptosis to eliminate irremediably damaged cells. To control cellular fate upon UPR induction, typically caused by overexpression of recombinant proteins, I designed a series of orthogonal genetic circuits that interface with the UPR, sense and integrate signals associated with activation of the UPR and, in response remodel the stress response pathway to enhance stress attenuation and delay apoptosis. Finally, I developed a nanobody-based platform for controlling the selective localization of a target protein to multiple cellular compartments and investigated different circuit architectures to obtain dynamic control over the localized target protein. Combining nanobody-mediated localization and degradation with orthogonal regulation, I generated a nanobody-based platform to control the relative concentration and localization of a target protein in cells. The tools described in this work are expected to have a significant impact on the design of strategies to reprogram mammalian cells for a diverse range of applications including the development of cell-based therapies and the production of biologics.
Embargo
Feedback-Regulated Cell Factories for Enhanced Therapeutic Protein Manufacturing
(2024-12-06) Barrios Santos, Daniela Maria; Segatori, Laura; Diehl, Michael; Chappell, James
The production of protein therapeutics is critical for maintaining a continuous supply of life-saving medications available in clinics. The production of large quantities of protein therapeutics, however, remains a major challenge in biomanufacturing. Overexpression of secretory proteins results in the accumulation of unfolded and misfolded off-pathway intermediates in the endoplasmic reticulum (ER), causing proteotoxic stress and, thus, affecting cell viability and protein productivity. Proteotoxic stress leads to the activation of the Unfolded Protein Response (UPR), a series of signal transduction pathways regulating protein quality control mechanisms aimed at restoring homeostasis. Sustained UPR activation culminates with the induction of apoptosis. Current strategies for enhancing the production of therapeutic proteins have focused on the deregulated modulation of key components of the UPR. These strategies have resulted in limited and often protein-specific improvements as they may lead to metabolic burden, disruption of homeostatic systems, and cell adaptation. Deregulated modulation of the UPR also does not account for the natural population heterogeneity characteristic of high protein expression systems. To address these limitations, I developed feedback-regulated cell factories that can sense proteotoxic stress and, in response, modulate the UPR to enhance stress attenuation and delay cell death. This work describes my efforts to engineer sophisticated genetic circuits that can interface with the innate signal transduction pathways of the UPR. To explore strategies for modulating the UPR in response to stress induced by overexpression of therapeutic proteins, I first investigated the dynamics of activation of the UPR signaling pathways mediating stress attenuation and apoptosis upon expression of different levels of a model secretory protein. I then developed a two-module system for modulating the UPR that consists of a UPR sensor and an actuator component. The UPR sensor was developed by genetically engineering cells to link the activation of an early marker of UPR stress to the expression of an orthogonal transcription factor, which translates the detection of UPR induction into activation of a genetic circuit mediating user-defined modulation of the UPR. I leveraged this cell engineering approach to generate three sense-and-respond systems designed to (1) enhance protein folding and secretion by amplifying the stress attenuation pathway of the UPR, (2) delay cell death by silencing the UPR-mediated pro-apoptotic response, and (3) combine amplification of stress attenuation and delay of apoptosis. I demonstrate that this cell engineering approach enabled dynamic UPR modulation upon induction of ER stress. I also show that combining stress attenuation with apoptosis delay enhanced the production of the therapeutic enzyme tissue plasminogen activator and the bispecific antibody blinatumomab. The feedback-responsive cell factories reported in this study are an innovative strategy to dynamically adjust the innate cellular capacity to cope with proteotoxic stress for enhancing therapeutic protein manufacturing.
From detection of proteasomal degradation to targeted control of protein depletion
(2017-07-28) Zhao, Wenting; Segatori, Laura
The ubiquitin proteasome system (UPS) regulates protein turnover and catalyzes degradation of misfolded or damaged proteins, thus playing an important role in maintaining protein homoeostasis in eukaryotic cells. The UPS has emerged as a drug target for diverse diseases characterized by altered protein homeostasis. However, while proteasome inhibitors are widely used in research and have transitioned to clinical settings, pharmacological agents that enhance proteasomal degradation are rare and poorly characterized. In addition, approaches aimed at controlling the expression levels of cellular proteins by modulating proteasomal degradation are typically based on cumbersome modifications of the target protein. To generate a tool to monitor increase in proteasomal degradation, I developed a genetic inverter (Deg-On system) that translates proteasomal degradation of a transcriptional regulator engineered to function as a UPS substrate into increase in expression of a fluorescent reporter (GFP), thereby linking enhancement of UPS activity to an easily detectable output. I demonstrate that this genetic inverter responds to modulation of UPS activity. Guided by predictive modeling, I modified this genetic inverter by introducing a positive feedback loop that allows self-amplification of the transcriptional regulator, thereby enhancing the output signal sensitivity and dynamic range. This technology for monitoring proteasomal activation will be useful for a variety of applications, including the discovery of UPS activators and the selection of cell lines with different levels of proteasome activity. To achieve fast, targeted, and predictable control of cellular protein levels without genetic manipulation of the target, I developed a technology for post-translational depletion based on a bifunctional molecule (NanoDeg) consisting of the antigen-binding fragment from the Camelidae sp. heavy-chain antibody engineered to mediate degradation through the proteasome. Guided by predictive modeling, I show that customizing the NanoDeg rate of synthesis, rate of degradation, or mode of degradation allows to achieve quantitatively predictable control over the target’s levels. Integrating a GFP-specific NanoDeg within the Deg-On system results in enhanced dynamic range and resolution of the output. By providing predictable control over cellular proteins’ levels, the NanoDeg could be used for systems-level analyses of cellular protein function and to improve the design of mammalian gene circuits.
Genetic and Chemical Activation of TFEB Mediates Clearance of Aggregated α-Synuclein
(Public Library of Science, 2015) Kilpatrick, Kiri; Zeng, Yimeng; Hancock, Tommy; Segatori, Laura; Bioengineering; Biosciences; Chemical and Biomolecular Engineering
Aggregation of α-synuclein (α-syn) is associated with the development of a number of neurodegenerative diseases, including Parkinson’s disease (PD). The formation of α-syn aggregates results from aberrant accumulation of misfolded α-syn and insufficient or impaired activity of the two main intracellular protein degradation systems, namely the ubiquitin-proteasome system and the autophagy-lysosomal pathway. In this study, we investigated the role of transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in preventing the accumulation of α-syn aggregates in human neuroglioma cells. We found that TFEB overexpression reduces the accumulation of aggregated α-syn by inducing autophagic clearance of α-syn. Furthermore, we showed that pharmacological activation of TFEB using 2-hydroxypropyl-β-cyclodextrin promotes autophagic clearance of aggregated α-syn. In summary, our findings demonstrate that TFEB modulates autophagic clearance of α-syn and suggest that pharmacological activation of TFEB is a promising strategy to enhance the degradation of α-syn aggregates.
Interplay of Perlecan and MMP-7/Matrilysin Regulates Metastatic Prostate Cancer Cell Behavior: Basic and Clinical Implications
(2015-04-23) Grindel, Brian John; Farach-Carson, Mary C; Lwigale, Peter; Segatori, Laura
Perlecan/HSPG2 is a large extracellular heparan sulfate proteoglycan concentrated at tissue borders and separating epithelium and stroma. Along with its proteolytic consumers, the matrix metalloproteinases (MMPs), perlecan helps orchestrate development and homeostasis in nearly all studied multicellular organisms. However, both molecule classes can be coopted by prostate cancer (PCa) to advance the disease to its most deadly metastatic form. This work aimed to understand that relationship both at the basic and clinical level. Perlecan with its HS chains and tight domain structure is generally resistant to proteolysis, but a PCa cell must produce an associated enzyme to cleave the border proteoglycan in order to metastasize. This work was the first to identify an active protease produced by PCa cells that can completely digest intact perlecan. Following in silico proteolytic analysis, matrilysin/MMP-7, was identified as a likely candidate for in vitro assays. MMP-7, unlike other enzymes tested, cleaved perlecan when presented in multiple contexts. Perlecan and a subdomain, domain IV-3 (Dm IV-3), but not other subdomains (Dm I, IV-1 and IV-2), induced a striking clustering phenotype. MMP-7 incubation completely reversed this effect to favor cell dispersion and adhesion. Proteomic signaling arrays point towards global Src kinase activation as a major influence of perlecan DmIV-3 effects. To determine if this perlecan/MMP-7 relationship exists in PCa subjects, I performed a tissue microarray, along with β2-microglobulin (β2M), a GF that binds perlecan and induces MMP secretion. Besides increased levels of the two proteins within the patients (cancer/normal), MMP-7 and perlecan levels statistically correlated in multiple grades and localized at tissue interfaces. Additionally, I developed a new assay probing the perlecan fragment signature in the same PCa subjects’ serum. Perlecan fragments were largely increased in PCa and some of the fragments were associated with MMP-7 expression in the subjects. Overall, this work demonstrates a unique interplay between perlecan and its efficient proteolyzer, MMP-7, a relationship that is relevant from the cell and tissue to the clinic and which is likely to contribute to PCa progression to metastatic lethal disease.
Methane Bioreforming for the Biosynthesis of Reduced Molecules at Ambient Pressure and Temperature
(2020-07-30) Crumbley, Anna Morgan; Gonzalez, Ramon; San, Ka-Yiu; Segatori, Laura
The inherent principles of biology offer access to low temperature and pressure reactions with high selectivity in single-reactor vessels as an efficient route to chemical production through value-added biomanufacturing. Remote methane (CH4), a low-cost, but high-energy, gaseous hydrocarbon comprises a potentially-attractive feedstock distributed globally and produced by both renewable and traditional sources. Utilizing CH4 as a feedstock for value-added biomanufacturing of products such as ammonia (NH3), an expensive but essential source of N in agriculture, would facilitate NH3 production in remote areas by addressing current technological challenges related to high temperature and pressure systems. This work details the development of a biological pathway producing NH3 from CH4, oxygen (O2), and nitrogen (N2) in the air at ambient pressure and temperature. A proof-of-concept system discussed herein demonstrates a syntrophic microbial consortium of Azotobacter vinelandii M5I3 and Methylomicrobium buryatense 5GB1 pAMR4-dtom1 performing methane bioreforming (MBR) and powering biological nitrogen fixation (BNF) through extracellular carbon and energy transfer. Using a co-culture of bacteria synthetically engineered to secrete extracellular electron carrier lactic acid and NH3 derived from CH4 and air, this work demonstrates accumulated extracellular carbon and nitrogen products in the proof-of-concept system. Through a combination of iterative refinement, control, and stable-isotope 15N2 labeling experiments, the work further demonstrates active BNF in the co-culture, albeit at a low level. The downstream portion of the pathway was optimized using in silico kinetic modeling tools and experimental synthetic biology modifications to enhance NH3 production by the system further. While overall NH3 yields remain low after implementing these modifications, in part due to gas-solubility mass transfer challenges, this work suggests that carbon and energy present in CH4 are capable of being utilized in a methane bioreforming manner to power enzymatic activity beyond carbon-based product generation. Furthermore, electron transfer to nitrogenase represents a bottleneck for efficient NH3 production in native biological nitrogen-fixing organisms.
Modeling a Reversed β-oxidation Cycle Into the Genome Scale Model of Zymomonas mobilis
(2013-09-16) Dash, Satyakam; Gonzalez, Ramon; Nagrath, Deepak; Segatori, Laura
This study proposes simulations which present optimized methods for producing fatty acids, fatty alcohols and alkanes using Zymomonas mobilis bacterium by the energy efficient β-oxidation reversal pathway, an eco-friendly alternative to the present petrol
Modulating Protein Homeostasis to Ameliorate Lysosomal Storage Disorders
(2012-09-05) Wang, Fan; Segatori, Laura; Bennett, George N.; Matthews, Kathleen S.; Zygourakis, Kyriacos
The goal of this project has been to develop therapeutic strategies for protein misfolding diseases caused by excessive degradation of misfolded proteins and loss of protein function. The focus for this work is lysosomal storage disorders (LSDs), a group of more than 50 known inherited metabolic diseases characterized by deficiency in hydrolytic enzymes and consequent buildup of lysosomal macromolecules. Gaucher’s Disease (GD) is used as a representative of the family of LSDs in this study. GD is caused by mutations in the gene encoding lysosomal glucocerebrosidase (GC) and consequent accumulation of the GC substrate, glucocerebroside. The most prevalent mutations among GD patients are single amino acid substitutions that do not directly impair GC activity, but rather destabilize its native folding. GC normally folds in the ER and trafficks through the secretory pathway to the lysosomes. GC variants containing destabilizing mutations misfold and are retrotranslocated to the cytoplasm for ER-associated degradation (ERAD). However, evidence shows that if misfolding-prone, mutated GC variants are forced to fold into their 3D native structure, they retain catalytic activity. This study describes strategies to remodel the network of cellular pathways that maintain protein homeostasis and to create a folding environment favorable to the folding of unstable, degradation-prone lysosomal enzyme variants. We demonstrated that folding and trafficking of mutated GC variants can be achieved by modulating the protein folding network in fibroblasts derived from patients with GD to i) upregulate the expression of ER luminal chaperones, ii) inhibit the ERAD pathway, and iii) enhance the pool of mutated GC in the ER amenable to folding rescue. We also demonstrated that the same cell engineering strategies that proved successful in rescuing the folding and activity of mutated GC enable rescue of mutated enzyme variants in fibroblasts derived from patients with Tay-Sachs disease, a LSD caused by deficiency of lysosomal hexosaminidase A activity. As a result, the current study provides insights for the development of therapeutic strategies for GD based on the modulation of general cellular pathways that maintain protein homeostasis that could in principle be applied to the treatment of multiple LSDs.
Modulating the Lysosome-Autophagy System to Restore Homeostasis in in vitro Model Systems of Lysosomal Storage Disorders
(2014-11-14) Song, Wensi; Segatori, Laura; Matthews, Kathleen; Gonzalez, Ramon
The protein quality control system is a complex network that promotes the folding and trafficking of newly synthesized proteins and regulates the degradation of misfolded proteins and protein aggregates. Failure of the quality control system to maintain protein homeostasis (or proteostasis) characterizes the cellular pathogenesis of a number of human diseases. In particular, this study focuses on lysosomal storage disorders, a group of inherited metabolic diseases characterized by deficiencies in specific lysosomal hydrolytic activities that result from mutations in genes encoding for lysosomal proteins and consequent buildup of lysosomal storage material. The ultimate goal of this work is to develop cell engineering strategies to modulate cellular quality control machineries that control protein folding, processing, and degradation to restore cellular homeostasis under conditions of proteotoxic stress. Specifically, this study aims to manipulate the lysosome-autophagy system to enhance folding and processing of lysosomal enzymes as well as to enhance the cellular clearance capacity. To achieve this goal, I investigated the role of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and function, in regulating lysosomal proteostasis and autophagic clearance. Specifically, chemical and genetic modulation of TFEB was found to enhance folding, trafficking and activity of unstable, degradation-prone lysosomal enzymes in in vitro models of lysosomal storage disorders. Moreover, pharmacologic activation of autophagy achieved by treating cells with 2-hydroxypropyl-β-cyclodextrin was found to enhance autophagic clearance of storage material specifically by activating TFEB. To further investigate the molecular mechanism of autophagy induction and activation of autophagic clearance, I tested the impact of polystyrene nanoparticles of different size and surface charge on the lysosome-autophagy system with the ultimate goal to link the physicochemical properties of nanomaterials with the specific nature of the autophagic response activated upon nanomaterial uptake into cells. Efficient autophagic clearance was found to depend highly on the surface charge. Specifically, cell exposure to polystyrene nanoparticles presenting neutral or negative surface charge results in activation of autophagic clearance, whereas cell exposure to polystyrene nanoparticles presenting cationic surface charge results in impairment of lysosomal function and blockage of autophagic flux. Ceria nanoparticles (or nanoceria) are widely used in a variety of applications including as UV blockers and catalysts in industrial processes. Recent studies also revealed that ceria nanoparticles present antioxidant properties, suggesting a potential role of nanoceria in a variety of biomedical applications. In this study, I investigated the impact of ceria nanoparticles stabilized by organic surface coatings on the lysosome-autophagy system, Ceria nanoparticles were found to activate the lysosome-autophagy system and enhance autophagic clearance. In summary, this work provides proof-of-principle demonstration of chemical and biological strategies to activate the lysosome-autophagy system for restoring lysosomal proteostasis and enhancing autophagic clearance in model systems of diseases characterized by deficiencies in lysosomal enzymes activities and aberrant accumulation of undegraded lysosomal substrates. These findings lay the foundation for the development of nanotherapeutics for the treatment of diseases associated with inefficient autophagic clearance.
Nanobody-mediated dynamic and spatial control of cellular proteins for mammalian gene circuit engineering
(2019-11-22) Kuypers, Brianna; Segatori, Laura
Cellular information processing in mammalian cells relies on sophisticated and highly dynamic mechanisms. Unravelling these mechanisms remains a challenging endeavor and has limited de novo design of biological functions. Controlling cellular phenotypes requires precise control of protein concentration and localization, which is typically realized through the design of genetic networks orthogonal to the cellular circuitry. This work describes my efforts to develop tools for achieving temporal and spatial control over cellular proteins through post-translational regulation that can be generally used to characterize and manipulate the complex genetic networks that regulate mammalian cells. I first illustrated the use of a nanobody-based platform designed to control degradation of target proteins (the NanoDeg) to improve the dynamic range and temporal resolution of input-dependent reporter systems. I then explored integration of a reporter-specific NanoDeg into a genetic circuit through a feedforward loop to generate an improved heat-shock reporter system. To enhance temporal control of genetic networks, I investigated strategies to use the NanoDeg for controlling the oscillatory behavior of a range of different circuit topologies. Finally, I developed a nanobody-based platform for controlling the subcellular localization of target proteins. Using this strategy, I experimentally demonstrated selective target localization to multiple cellular compartments and investigated different circuit architectures for dynamic control of target localization to different compartments. Combining nanobody-mediated localization and degradation with orthogonal transcriptional regulation I developed a nanobody-based toolkit to achieve spatial and temporal control of target protein levels.
Novel enzymes and pathways facilitating biological utilization of one carbon compounds
(2018-11-30) Chou, Alex; Gonzalez, Ramon; Segatori, Laura; Phillips, George
Although biological systems hold great potential for the sustainable production of fuels and chemicals from one-carbon (C1) feedstocks (1), their C1 utilization reactions rely on specific acceptor molecules, complex metabolic pathways, and originate from difficult to engineer microorganisms, limiting applicability and implementation in the biotechnology industry (2–4). As a result, to date no non-native C1 utilizing organism has been engineered for growth or product synthesis from solely C1 substrates. In this thesis, we report that 2-hydroxyacyl-CoA lyase (HACL), an enzyme involved in the α-oxidation of long-chain fatty acids, catalyzes a novel C1 addition reaction resulting in the ligation of carbonyl-containing molecules with formyl-CoA to produce C1-elongated 2-hydroxyacyl-CoAs. We characterized the first prokaryotic variant of HACL and found that it can use a wide range of carbonyl substrates of different chain lengths, including both aldehydes and ketones, which when combined with enzymes comprising a de novo designed pathway, supported the conversion of C1 feedstocks to industrially relevant chemicals such as glycolate, ethylene glycol, ethanol, acetate, glycerate, and 2-hydroxyisobutyrate. Homology-guided mutagenesis allowed the identification of key residues influencing the in vivo activity of HACL and its ability to support C1 bioconversion. We implemented an HACL-based pathway in E. coli to utilize formaldehyde as the sole carbon substrate for glycolate production and demonstrated the potential of the pathway to support growth in a two-strain system, which was supported by genome scale modeling and flux balance analysis. The previously undescribed condensation reaction catalyzed by HACL is a direct and flexible means for C1 addition that can facilitate engineering of C1 bioconversion and synthetic methylotrophy/autotrophy in industrial organisms.
Open questions: how do engineered nanomaterials affect our cells?
(BioMed Central, 2020) Barrios, Daniela; Segatori, Laura; Bioengineering; Biosciences; Chemical and Biomolecular Engineering
Our cells have evolutionarily conserved mechanisms that battle foreign and toxic materials to maintain cellular homeostasis and viability. How do these cellular machineries respond to engineered nanomaterials?
Optimization of Proteasomal Degradation Reporter (eDeg-On) System for CRISPR-mediated Whole-genome Knockout Screens
(2017-04-21) Liu, Yiwen; Segatori, Laura; Zhong, Weiwei
Protein folding and clearance of misfolded proteins are crucial to maintain cellular homeostasis (Jariel-Encontre et al., 2008). Misfolded proteins may associate with other cellular components and possibly impair their functions. They may also self-associate to form insoluble aggregates, which are the hallmarks of a number of neurodegenerative diseases, such as Parkinson’s (Olanow and McNaught, 2006) and Alzheimer’s (Oddo, 2008). The ubiquitin proteasome system (UPS) is the main pathway that catalyzes the degradation of soluble misfolded proteins in mammalian cells. Therefore, enhancing the UPS activity through activation of proteasomal degradation is considered a promising strategy to ameliorate phenotypes associated with the accumulation of misfolded proteins. Modulation of specific UPS components, for instance, results in increased degradation of target proteins (Rechsteiner and Hill, 2005 and Vilchez et al., 2012). However, our current understanding of the molecular mechanism underlying proteasomal degradation is still limited, limiting the rational design of pharmacologic strategies to enhance UPS activity. As a result, proteasome activators are rare and remain poorly characterized (Huang and Chen, 2009). To overcome these limitations, researchers in my group developed a cell-based platform (the eDeg-On system) to monitor changes in UPS activity. This genetic circuit links increase in UPS activity to an increase in fluorescent output, thereby providing a reliable tool for the discovery of proteasome activators. The CRISPR-cas technology has emerged as powerful technique to introduce genetic modifications at the whole-genome scale. I optimized the eDeg-On system and evaluated it for pooled screening of whole-genome CRISPR-mediated knockout library. I replaced the antibiotic resistance gene in the eDeg-On system and assessed the response of HEK293 cells stably expressing the eDeg-On system to modulation of proteasomal degradation. To evaluate the use of a stable cell line expressing the eDeg-On system as a reporter assay in the context of a pooled CRISPR-mediated screen, I conducted mock screens using different ratios of positive and negative controls. The results obtained demonstrate that the eDeg-On system can be used as a reporter assay for CRISPR-mediated whole-genome knockout screens. The use of the eDeg-On system to conduct genetic screen for the discovery of molecules that function as proteasome regulators will contribute to the development of therapeutic strategies for protein misfolding diseases. Further applications of this study include targeting the UPS function for therapeutic applications as well as for enhancing the production of recombinant proteins in industrial settings.

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