Browsing by Subject "Molecular biology"

Osteoporosis and low bone density affect an estimated 54 million adults of 50 years and over in the United States, resulting in $19 billion in costs for osteoporosis-related bone breaks. Current treatments include the use of antiresorptive and anabolic drugs to decrease the rate of bone resorption and increase the rate of bone formation, respectively. However, these current treatments are unable to completely normalize skeletal integrity. As bone diseases become increasingly prevalent, there is an urgent need to identify novel therapies to improve quality of life and reduce economic burden on the healthcare system.

To identify novel therapeutic targets, we must first begin to understand the cellular complexity of the bone marrow niche and how cellular function is regulated within the bone tissue. Bone-resident cells, such as skeletal progenitors and their descendants, are critically influenced by extrinsic signals derived from the local microenvironment. Previous studies have identified hypoxia as a key microenvironment factor in bone. Thus, the ability to target the hypoxic bone marrow niche presents an attractive and untapped potential for regenerative medicine.

Much of the work investigating the role of hypoxia and HIF signaling have focused on mature osteoblast and chondrocyte populations. In contrast, studies investigating the contribution of HIF signaling on skeletal progenitors and marrow adipocyte populations are scarce. In this dissertation, I investigate the role of hypoxia and HIF signaling in skeletal lineage cells, chiefly skeletal progenitor cells and marrow adipogenic lineage cells. Using cellular, genetic, and pharmacological-based approaches, I characterize the roles of HIF-1α and HIF-2α in both homeostatic and pathological contexts in the aforementioned cell populations.

First, I propose an optimized cell-based system to investigate the function of skeletal progenitors in vitro. Here, I highlight the limitations of current in vitro isolation techniques and introduce a relatively simple method of bone marrow stromal cell purification using hypoxia. Using this system, I assess how skeletal progenitors respond to hypoxic cues and interrogate skeletal progenitor cell differentiation and functional responses in my subsequent research. Next, using genetic and pharmacological approaches, I investigate the role of HIF-2α in bone formation following radiation-injury where I identify HIF-2α as a negative regulator of bone recovery. Additionally, with the assistance of my collaborators, I develop and characterize a bone-targeting nanocarrier to ameliorate radiation-induced bone loss. Lastly, I detail early work I conducted to investigate the role of HIF signaling in marrow adipogenic lineage cells. Here, I establish and characterize animal models to determine how hypoxia and HIF signaling influences adipogenic lineage commitment and expansion in an early and mature marrow adipogenic population.

In summary, this dissertation aims to expand our limited understanding on how the hypoxic bone microenvironment and HIF signaling regulate skeletal lineage cells in vivo, with a special focus on skeletal progenitor and marrow adipogenic populations. In terms of boarder impacts, understanding the signaling networks that regulate bone homeostasis and recovery processes will not only expand our basic understanding of the molecular mechanisms underlying skeletal development, but also provide novel insights for developing therapies to treat bone loss.

Basement membrane (BM) is a sheet-like extracellular matrix that underlies most tissues and acts as a barrier to invading cells. Many cell types, including immune cells, cells migrating during development and morphogenesis, and metastatic cancer cells utilize F-actin-based structures called invadopodia to breach BM as they leave one tissue to enter another. Despite extensive study and interest in understanding invasion for its clinical importance, the molecular mechanisms regulating invadopodia and BM breach in vivo remain unclear. During uterine-vulval attachment in C. elegans, the specialized uterine anchor cell (AC) uses invadopodia to mediate breach of the underlying BM in order to contact the underlying vulval epithelium. The AC offers several advantages as a model, including experimental, visual, and genetic tractability, the presence of endogenous extracellular environment, and availability of the tissue targeted for invasion. In Chapter 2, I describe development of a novel technique for spatiotemporal-specific knockdown of proteins that will facilitate investigation of proteins, particularly those that are essential or required in other tissues, in the regulation of invadopodia and AC invasion. Using a sensitized genome-wide RNAi screen, classical genetics, and timelapse imaging of invadopodia at the AC-BM interface, Chapter 3 presents two in vivo invadopodia regulators that function by distinct mechanisms. This is the first in vivo evidence that the RhoGTPase CDC-42 regulates invadopodia formation through WSP-1. RabGTPase GDI-1 is a novel regulator of the unique membrane compartment required for invadopodia formation. CDC-42 and GDI-1 both function downstream of an unknown cue secreted by the cells targeted by the AC for invasion, illustrating that extracellular cues can play key roles in mediating cell invasion. The characterization of CDC-42 and GDI-1 as in vivo regulators of invadopodia is an important first step to understanding the mechanisms of this critical cellular process and we expect the AC will be an excellent model for future identification of novel regulators of BM breach.

During the course of breast cancer progression, normally dormant tumor-promoting effects of transforming growth factor-beta (TGF-beta) including migration, invasion, and metastasis are unmasked. Although this switch or gain of TGF-beta function has been modeled extensively in in-vivo and in-vitro breast cancer systems, the signaling mechanisms that control this TGF-beta switch are poorly understood. Indeed, the precise role of canonical TGF-beta signaling through the type I TGF-beta receptor, ALK5, and its intracellular effectors, Smad2 and Smad3, is still poorly understood. In an effort to identify mechanisms that regulate the ability of TGF-beta to stimulate mammary epithelial cell migration in-vitro, we found that TGF-beta stimulates the phosphorylation of Smad1 and Smad5, intracellular effectors that are typically associated with bone morphogenetic protein (BMP) signaling. As this phosphorylation response has not been reported extensively, little is known about the prevalance, mechanism, function, or pathological relevance of TGF-beta-stimulated Smad1/5 phosphorylation.

Herein, we use pharmacologic inhibition, RNA interference, and additional biochemical and cell-based approaches to identify a novel mechanism and function for non-canonical TGF-beta signaling through an ALK5-Smad1/5 axis. We show that TGF-beta stimulates Smad1/5 phosphorylation in an ALK5 dependent manner in cells of epithelial, endothelial, and embryonic origin. Mechanistically, this phosphorylation event requires the kinase activity and, unexpectedly, the L45 loop motif of ALK5. Functionally, this phosphorylation event is essential to the initiation and promotion of TGF-beta-stimulated migration in mammary epithelial cells. Interestingly, this phosphorylation event may promote migration by regulating TGF-beta target gene expression, as evidenced by the identification of putative Smad1/5-dependent TGF-beta target genes using microarray analysis. Finally, of particular relevance to mammary tumor progression, this phosphorylation event is preferentially detected in permissive environments such as those created by tumorigenic cells or HER2 oncogene activation.

Taken together, our data provides evidence that TGF-beta-stimulated Smad1/5 phosphorylation, which occurs through a non-canonical mechanism that challenges the notion of selective Smad phosphorylation by ALK5, mediates the pro-migratory TGF-beta switch in mammary epithelial cells.

Regulation of cell growth is controlled by a variety of factors, including a number of oncogenes and tumor suppressors. PTEN is an inositol phosphatase that regulates cell growth by hydrolyzing the phospholipid products of PI3K. PTEN is mutated in a number of cancers, leading to its characterization as an important tumor suppressor. Recent data indicate that PTEN may also perform important functions that are independent of its phosphatase activity, most notably within the nucleus. Studies in this thesis addressed a novel role for PTEN in the regulation of the cellular response to hypoxia.

PTEN overexpression significantly increased hypoxic gene expression independent of its catalytic activity, while shRNA-mediated silencing of PTEN significantly inhibited hypoxia-mediated HRE-luciferase activity. Nuclear-localized PTEN was more effective in promoting HRE activity than nuclear-excluded PTEN. These results suggested a scaffolding function of PTEN in the hypoxic nucleus. To identify specific gene targets regulated by PTEN in hypoxia, a custom oligo-array consisting of 46 hypoxia-responsive genes was utilized following both gain- and loss-of- PTEN function. Based on real-time quantitative results, PTEN positively regulated genes involved in metabolism (PFKFB3, PFKFB4, ALDOA, PGK-1), oxygen supply (VEGFA, EPO), cell growth (Tgf-a, TERT, cyclin D1, BNIP3), motility (E-cadherin) and transcription (DEC2). A single missense mutation at isoleucine 224 (I224M) of PTEN, however, abrogated the ability of PTEN to regulate the hypoxia response without affecting its lipid phosphatase activity. PTEN has previously been shown to bind to the co-activator p300 and to affect p53 acetylation and stabilization. As p300 is also a co-activator for the HIF proteins, we hypothesized that PTEN's association with p300 would promote the HIF/p300 complex to positively regulate hypoxic gene transcription. Overexpression of PTEN-WT extended the half-life of p300 and histone acetyltransferase activity of p300 in hypoxia, while overexpression of PTEN-I224M or PTEN silencing decreased both. In vivo, these effects resulted in a significant increase in hypoxic area in PTEN-null tumors compared to tumors expressing endogenous levels of PTEN, suggesting an inability to mount a hypoxia response necessary for revascularization of the tissue. PTEN's effect on p300 extended to other functions of p300 outside of the hypoxia response, most notably p300's role in p53 stability and p53-mediated gene transcription. Overexpression of PTEN resulted in an increase in p53 reporter activity following DNA damage (mitomycin C treatment). PTEN silencing or overexpression of PTEN-I224M resulted in abrogation of these effects. Taken together, these findings demonstrate that PTEN is required for the hypoxia response and they suggest that PTEN acts as a scaffold for p300 and the HIF machinery in the hypoxic nucleus independent of its canonical lipid phosphatase activity. These results may have important implications for the treatment of tumors in which PTEN is lost or mutated. The potential use of PTEN-I224M as a therapeutic is also discussed

Bisphosphate 3'-nucleotidase (Bpnt1) is a member of a family of small molecule phosphatases whose activities depend on divalent cations and are inhibited by lithium. While the enzymes share many commonalities, they have distinct and non-­overlapping substrate pools. Of the seven mammalian members, two enzymes, gPAPP and Bpnt1, hydrolyze the same small molecule 3'-phosphoadenosine 5'-phosphate (PAP) but act in separate subcellular compartments, the Golgi apparatus and cytoplasm respectively. Hydrolysis of PAP, which is a metabolite of the inorganic sulfate incorporation pathway, is highly conserved throughout evolution from bacteria to yeast to humans. Evidence in multiple species has shown that inhibiting PAP hydrolysis leads to cellular toxicity as a result of its accumulation and also that these effects can be ameliorated by modulating the rate of its production. However, despite the abundant evidence of its importance

from studies in lower eukaryotes, the role of the cytoplasmic PAP phosphatase, Bpnt1, in more complicated mammalian physiological remains poorly understood. Here we report for the first time the generation and characterization of mice deficient for Bpnt1. Bpnt1 null mice do not exhibit skeletal defects, but instead develop severe liver

pathologies and deficiencies in intestinal iron absorption. Loss of Bpnt1 leads to tissue-specific elevations of the substrate PAP. To test the hypothesis that a toxic cellular accumulation of PAP accounts for the observed phenotypes, we generated a double mutant mouse that concomitantly down regulates bisphosphorylated nucleotide synthesis in the context of Bpnt1 deficiency. Remarkably, double mutants do not display any detectable physiological defects seen in Bpnt1 null mice. In addition, we have identified and characterized a novel substrate of 3'­nucleotidases, 3'-phosphoadenosine 5'-diphosphate (PAPP) that co-accumulates with PAPS and PAP and might play a role in mediating certain aspects of the physiological defects of Bpnt1 null mice. Overall, our study defines a role for Bpnt1 in mammalian physiology and provides mechanistic insights into the importance of cytoplasmic 3'-­

nucleotide hydrolysis to normal cellular function.

The Rho GTPase Cdc42 is a master regulator of cell polarity that orchestrates reorganization of the cytoskeleton. During polarity establishment, active GTP-Cdc42 accumulates at a part of the cell cortex that becomes the front of the cell. Localized GTP-Cdc42 orients the cytoskeleton through a set of “effector” proteins that bind specifically to GTP-Cdc42 and not GDP-Cdc42. A family of Cdc42 effectors, called GICs in yeast and BORGs in mammals, have been implicated in regulation of both the actin cytoskeleton and the septin cytoskeleton. Yeast cells lacking both Gic1 and Gic2 are able to polarize and grow at low temperatures, but many mutant cells fail to polarize the cytoskeleton at high temperature. This led to the conclusion that GICs communicate between Cdc42 and different cytoskeletal elements.

To better characterize the role of GIC proteins in yeast, we utilized time-lapse fluorescent microscopy to examine morphogenetic events in living single cells. Surprisingly, we found that not only the cytoskeleton but also Cdc42 itself failed to polarize in many gic1 gic2 mutant cells at high temperature. This observation indicates that GICs may act upstream of polarization rather than downstream.

Polarization of Cdc42 is triggered by cell-cycle progression, and in particular by G1 Cyclin-dependent kinase (CDK) activity. Using a live-cell reporter for G1 CDK activation, we found that cells lacking GICs were not defective in CDK activation, but showed a specific defect in polarization downstream of the CDK. Previous work had implicated the scaffold protein Bem1 in a positive feedback loop important for polarization. Cells lacking GICs failed to polarize Bem1 as well as Cdc42 at high temperature. Future work will be directed at understanding how GICs contribute to polarity establishment. Because many of the mechanisms and proteins involved in polarization are highly conserved, we anticipate our findings will help inform how this process regulated in higher eukaryotes.

Transforming growth factor–β (TGF–β) signaling regulates a range of processes in a variety of cell types. Consequently, TGF–β plays a complex role in the progression of several types of cancers; it acts as a tumor suppressor in normal cells and early in tumor progression, yet it can promote tumor progression in later stages of cancer.

Among the cancers that TGF–β has been implicated in is glioblastoma multiforme (GBM), the most common primary brain neoplasm and one of the most lethal types of cancer. Because of its high mortality rate and the lack of effective treatments, discovering the molecular mechanisms that underlie GBM formation and growth is of great clinical interest. To this end, we investigated the function of a TGF–β target gene — the putative tumor suppressor N‐Myc downstream‐regulated gene 4 (NDRG4) — in GBM cell viability, proliferation and tumor formation. Contrary to the established roles of other NDRG family members, we found that NDRG4 expression is elevated in GBM and that NDRG4 is required for the survival of established GBM cell lines and primary GBM xenograft cells enriched for highly tumorigenic GBM cancer stem cells. Knockdown of NDRG4 expression results in G₁ cell cycle arrest followed by apoptosis that is associated with a decrease in the expression of XIAP and survivin. Finally, knockdown of NDRG4 expression in established GBM cell lines and GBM cancer stem cells results in decreased tumorigenicity following intracranial implantation of these cells into immunocompromised mice. Collectively, these data indicate that NDRG4 does not function as a tumor suppressor like other NDRG family members, but rather it is essential for GBM tumorigenicity and may represent a potential therapeutic target for this devastating disease.

In the second portion of this dissertation, we examine the TGF–β cytostatic signaling pathway in B lymphocytes. TGF–β–induced growth inhibition is the most extensively studied biological response to a TGF–β signal. Although in most cell types this response is mediated by Smad3– dependent regulation of c–Myc, p15Ink4B, and p21Cip1 transcription, studies from Smad3 null mice suggest that TGF–β–induced growth inhibition in B lymphocytes occurs regardless of Smad3 status. We prove that this response does indeed occur independently of Smad3 in purified primary B lymphocytes and WEHI–231 cells. Consistent with this, p15Ink4B and p21Cip1 are not noticeably induced by TGF–β in these cells, whereas Id3 and cyclin G2 are induced in a Smad3–independent manner. Finally, unlike the MAPK pathways we tested, the BMP–specific Smads 1 and 5 are activated in response to TGF–β in these cells, and this activation is dependent on ALK5 kinase activity. Collectively, these data indicate that TGF–β induces growth inhibition in B lymphocytes through a novel signaling pathway, and Smads 1 and 5 may help mediate this response.

The vasculature has a crucial function in normal physiology, enabling the transport of oxygen and nutrients to cells throughout the body. In turn, endothelial cells, which form the inner-most lining of blood vessels, are key regulators of vascular function. In addition to forming a barrier which separates the circulation from underlying tissues, endothelial cells respond to diverse extracellular cues and produce a variety of biologically-active mediators in order to maintain vascular homeostasis. Disruption of normal vascular function is a prominent feature of a variety of pathological conditions. Thus, elucidating the signaling pathways regulating endothelial function is critical for understanding the role of endothelial cells in both normal physiology and pathology, as well as for potential development of therapeutic interventions.

In this dissertation, we use a combination of pharmacological inhibition and knockdown studies, along with generation of endothelial conditional knockout mice, to demonstrate an important role of the Abelson (Abl) family of non-receptor tyrosine kinases (Abl and Arg) in vascular function. Specifically, loss of endothelial expression of the Abl kinases leads to late-stage embryonic and perinatal lethality in conditional knockout mice, indicating a crucial requirement for Abl/Arg kinases in normal vascular development and function. Endothelial Abl/Arg-null embryos display focal regions of vascular loss and tissue damage, as well as increased endothelial cell apoptosis. An important pro-survival function for the Abl kinases is further supported by our finding that either microRNA-mediated Abl/Arg depletion or pharmacological inhibition of the Abl kinases increases endothelial cell susceptibility to stress-induced apoptosis in vitro. The Abl kinases are activated in response to treatment with the pro-angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). We show that both VEGF- and bFGF-mediated endothelial cell survival is impaired following Abl kinase inhibition.

These studies have uncovered a previously unappreciated role for the Abl kinases in the regulation of the angiopoietin/Tie2 signaling pathway, which functions to support endothelial cell survival and vascular stability. Loss of Abl/Arg expression leads to reduced mRNA and protein levels of the Tie2 receptor, resulting in impaired activation of intracellular signaling pathways by the Tie2 ligand angiopoietin-1 (Angpt1), as well as decreased Angpt1-mediated endothelial cell survival following serum-deprivation stress. Notably, we found that the Abl kinases are activated following Angpt1 stimulation, suggesting a unique dual role for Abl and Arg in Angpt/Tie2 signaling, potentially modulating Tie2 downstream signaling responses, as well as regulating Tie2 receptor expression.

Further, we show an important contribution of the Abl family kinases to the regulation of endothelial permeability responses both in vitro and in vivo. The Abl kinases are activated in response to a diverse group of permeability-inducing factors, including VEGF and the inflammatory mediators thrombin and histamine. We show that inhibition of Abl kinase activity, using either the ATP-competitive inhibitor imatinib or the allosteric inhibitor GNF-2, protects against disruption of endothelial barrier function by the permeability-inducing factors in vitro. VEGF-induced vascular permeability similarly is decreased in conditional knockout mice lacking endothelial Abl expression, as well as following treatment with Abl kinase inhibitors in vivo. Mechanistically, we show that loss of Abl kinase activity is accompanied by activation of the barrier-stabilizing GTPases (guanosine triphosphatases) Rac1 and Rap1, as well as inhibition of agonist-induced Ca2+ mobilization and generation of acto-myosin contractility.

Taken together, these results demonstrate involvement of the Abl family kinases in the regulation of endothelial cell responses to a broad range of pro-angiogenic and permeability-inducing factors, as well as a critical requirement for the endothelial Abl kinases in normal vascular development and function in vivo. These findings have implications for the clinical use of Abl kinase inhibitors.

Adherens junctions are calcium-dependent cell-cell contacts formed during epithelial morphogenesis that link neighboring cells via cadherin receptors. Coordinated regulation of the actin cytoskeleton by the Rho GTPases is required for the formation and dissolution of adherens junctions, however the pathways that link cadherin signaling to cytoskeletal regulation remain poorly defined. The Abl family of tyrosine kinases have been shown to modulate cytoskeletal reorganization downstream of various extracellular signals including growth factor receptors and integrins.

Here we use pharmacological inhibition and RNA interference to identify the Abl family kinases as critical mediators of cadherin-mediated adhesion. Endogenous Abl family kinases, Abl and Arg, are activated and are required for Rac activation following cadherin engagement, and regulate the formation and maintenance of adherens junctions in mammalian cells. Significantly, we show that Abl-dependent regulation of the Rho-ROCK-myosin signaling pathway is critical for the maintenance of adherens junctions. Inhibition of the Abl kinases in epithelial sheets results in activation of Rho and its downstream target ROCK, leading to enhanced phosphorylation of the myosin regulatory light chain. These signaling events result in enhanced stress fiber formation and increased acto-myosin contractility, thereby disrupting adherens junctions. Conversely, Arg gain-of-function promotes adherens junction formation through a Crk-dependent pathway in cells with weak junctions. These data identify the Abl kinases as a novel regulatory link between the cadherin/catenin adhesion complex and the actin cytoskeleton through regulation of Rac and Rho during adherens junction formation.

Unexpectedly, we identified a requirement for Abl and Crk downstream of Rac in the regulation of adherens junctions. Therefore, Abl functions both upstream and downstream of Rac in regulating adherens junctions, which suggests the possibility of a positive feedback loop consisting of Abl-Crk-Rac.

Finally, we identified the Abl kinases as critical mediators of epithelial cell response to HGF. Pharmacological inhibition of Abl kinase activity resulted in impaired dissolution of adherens junctions downstream of HGF stimulation of the Met receptor. Additionally, we observed decreased phosphorylation of the Met receptor itself, along with Gab1 and Crk, downstream effectors of Met signaling. Taken together, these data suggest a requirement for Abl kinases in both adherens junctions formation and turnover.

Current standard cancer treatments such as chemotherapeutics, and radiation therapy are nearly as likely to kill the patient as cure the cancer. Therapies that have such a narrow window of efficacy are necessary for the treatment of aggressive diseases, but safer alternatives must be created. By discovering novel therapeutics that target specific disease processes within specific diseased cells, while leaving healthy cells unaltered, we can improve the lives of millions of cancer sufferers and their families. A therapeutic's window of efficacy can be measured by the therapeutic index. For many anti-cancer therapeutics, the therapeutic index is very small, the dose of treatment that kills cancer cells and shrinks tumors is nearly the dose that causes toxicity. In cancer patients, this toxicity causes many serious conditions such as gastrointestinal distress, organ damage, and death.

Recently, the model of cancer treatment has evolved from non-specific cytotoxic agents to more selective therapeutics that target cellular processes necessary for cancer cell survival. If a therapy can be targeted to selectively bind and internalize targeted cells, its toxicity would only impact the targeted cells and healthy cells in the immediate vicinity, which would greatly reduce the toxic effects on the rest of the body. Targeting cancer cells can be done through cancer biomarkers, which are cell surface proteins, expressed exclusively, or are much more abundant on the surface of cancer cells than on somatic cells.

Advances in antibody and aptamer technology have enabled researchers to design those molecules to bind specifically to cancer cells, and deliver drugs that alter specific cellular processes. An aptamer designed to bind PSMA, a prostate cancer biomarker, only bound to a specific subset of cancer cells, and delivered a therapeutic siRNA that prevented a specific survival process from occurring. While this technology is promising, it is currently limited to targeting small subsets of cancer types. To generate an aptamer therapeutic that would have greater utility and efficacy, I have examined the properties of a nucleolin aptamer-mediated delivery system that targets multiple types of cancer cells, and delivers various oligonucleotide therapeutics.

The nucleolin aptamer targeted cancer cells by binding to membrane–associated nucleolin. Nucleolin, a conserved protein found in all eukaryotes, shuttles from the nucleus, through the cytoplasm to the cell membrane. Cancer cells express a far greater amount of membrane–associated nucleolin than somatic cells, making nucleolin an ideal cancer biomarker. The shuttling, and oligonucleotide binding attributes of the protein enable it to deliver aptamer chimeras from the cell surface to the nucleus. Therefore the nucleolin aptamer has unique access to the nuclei of cancer cells, and can deliver therapeutic oligonucleotide cargoes through nucleolin binding.

The nucleolin aptamer delivered splice–switching oligonucleotides, a form of antisense technology, improving their efficacy, and potentially increasing their therapeutic viability. The ability to deliver antisense oligonucleotides to the nuclei of cancer cells has the potential for other therapeutic possibilities including the inhibition of transcription with antisense triplexes.

The nucleolin aptamer can also deliver therapeutic aptamers. The nucleolin aptamer–β–arrestin aptamer chimera prevented the stem cell renewal phenotype necessary for leukemia progression in human patient tissue samples. The ability to effectively deliver therapeutic aptamers may lead to clinical applications for many of the aptamers that have been selected against intracellular targets including transcriptional activators.

Oligonucleotide research continues to advance our understanding of potentially therapeutic oligonucleotides. Long non–coding RNAs for example, may impact epigenetics, and transcription. Additionally, locked nucleic acids have been developed to improve binding affinity, thus increasing the efficacy of antisense oligonucleotides. In order to bring these discoveries into the clinic, they must be safely and specifically delivered to their target cells.

This work demonstrated that the nucleolin aptamer could deliver oligonucleotide therapeutics to specific cancer cells. Nucleolin aptamer chimeras have the potential to develop into safe and effective cancer therapies, thus improving the treatment options for cancer sufferers.

The homeostatic hematopoietic stem cell compartment is comprised of quiescent long term self renewing stem cells and cycling short term stem cells with finite renewal potential. To study the molecular mechanisms governing self renewal of hematopoietic cells we must force them to enter the cell cycle and proliferate. One approach to accomplish this goal is to damage the hematopoietic compartment with ionizing radiation or cytotoxic chemotherapy. Such injuries ablate mature blood cells and drive the primitive stem cells into cycle. I have elected to use a simple model of hematopoietic damage and regeneration to study the molecular mechanisms controlling self renewal in hematopoietic stem cells. At the beginning of this project it was unclear whether the signaling pathways which homeostatically control self renewal are utilized during injury repair. In particular, there is very little understanding of the signals required for regeneration after radiation damage. We hypothesized extracellular signal transduction pathways provided by the microenvironment are critical mediators of the stem cell repair process. To address these topics and extend the previous work generated in our laboratory, I chose to pursue a candidate approach focusing on the Wnt and Notch developmental signaling pathways.

In order to examine the activation and requirement for each signaling cascade after radiation and chemotherapy damage we used a combination of loss of function and reporter mouse models. To this end, we have conducted the majority of experiments for the Wnt project in animals deficient in beta-catenin, the key transcription factor required in the pathway. Our investigations revealed the Wnt pathway is turned on within regenerating stem cells and loss of beta-catenin impairs regeneration of the stem cell compartment after both radiation and chemotherapy injury.

Using a Transgenic Notch Reporter mouse to investigate the role of Notch signaling following hematopoietic damage we determined the Notch pathway is also activated during regeneration. Furthermore, using a live imaging approach we discovered Notch activated cells change their fate choice during regeneration. To determine if Notch gain of function provides radio-protection we infected stem cells with an active form of Notch prior to radiation and then scored self renewal potential in vitro. This led us to the conclusion that Notch gain of function can provide a self renewal benefit to irradiated hematopoietic stem cells.

Astrocytes, the perisynaptic glial cells of the brain, display a complex morphology that is strongly linked to their functions at the synapse. Primary processes radiating from the astrocyte cell soma branch out to secondary and tertiary processes, which further ramify into tiny perisynaptic astrocyte processes, giving a mature astrocyte its characteristic arborized structure. Astrocyte processes dynamically ensheath the pre- and post-synapse to provide instructive cues for synapse formation, maturation, and function. Perturbations in astrocyte-synapse interactions result in synaptic deficits, leading to excitation/inhibition imbalance and aberrant neural circuitry. However, the mechanisms linking astrocyte morphology and function to neuronal contact and synaptic adhesion are poorly understood. In a candidate-based reverse genetic screen utilizing rodent cortical neurons and astrocytes, I identified two genes, HepaCAM and CTNND2, as regulators of astrocyte morphogenesis in response to neuronal adhesion.HepaCAM is an astrocyte-enriched cell adhesion molecule that participates in cell-cell and cell-ECM interactions to regulate cell migration and proliferation. shRNA-mediated silencing of hepaCAM expression in astrocytes resulted in decreased astrocyte complexity in vitro and in vivo. HepaCAM stabilizes the gap junction protein connexin 43 (Cx43) at cell-cell junctions. We used stimulated emission depletion (STED) microscopy to show that hepaCAM and Cx43 colocalize at astrocyte processes in the mouse cortex and performed native affinity purifications followed by liquid chromatography-coupled high-resolution mass spectrometry (AP-MS) to demonstrate that Cx43 binds to hepaCAM. Finally, utilizing the same shRNA silencing approach, we found that hepaCAM and Cx43 were epistatic to each other in the regulation of astrocyte morphogenesis. Through mosaic analysis with double markers (MADM), we found that hepaCAM knockout astrocytes lost their ability to tile and had mislocalized Cx43. Consequently, gap junction coupling is impaired in astrocytes without hepaCAM. Additionally, we found decreased colocalization of hepaCAM puncta with synapses, a marked decrease in inhibitory synapses density, and a significant decrease in amplitude of miniature inhibitory postsynaptic currents, suggesting that loss of astrocytc hepaCAM disrupts the balance between synaptic excitation and inhibition. During development, astrocytes need to form non-overlapping territories within which they dynamically ensheathe synapses within discrete regions of neuropil. Taken together, our findings suggest that hepaCAM and Cx43 are critical proteins at the intersection of these two processes to ensure the proper molecular regulation of astrocyte self-organization and territory formation for normal circuit formation and function. Next, we identified Ctnnd2 (protein: δ-catenin) as another key regulator of astrocyte morphological complexity. δ-catenin was previously thought to be a neuron-specific protein that regulates dendrite morphology. Utilizing RNA fluorescence in situ hybridization (RNA-FISH) and immunohistochemistry, we found Ctnnd2 mRNA and δ-catenin is also highly expressed by astrocytes during the critical period of astrocyte morphological maturation and synapse formation during cortical development. shRNA-mediated silencing of Ctnnd2 expression in astrocytes resulted in decreased astrocyte complexity in vitro and in vivo. δ-catenin is hypothesized to mediate transcellular interactions through the cadherin family of cell adhesion proteins. We used structural modeling and surface biotinylation assays in both HEK293T and purified astrocyte cultures to reveal that δ-catenin interacts with N-cadherin juxtamembrane domain to promote N-cadherin surface expression. An autism-linked δ-catenin point mutation impaired N-cadherin cell surface expression and reduced astrocyte complexity. In the developing mouse cortex, only lower-layer cortical neurons express N-cadherin. Remarkably, when we silenced astrocytic N-cadherin throughout the cortex, only lower-layer astrocyte morphology was disrupted. These findings show that δ-catenin controls astrocyte-neuron cadherin interactions that regulate layer-specific astrocyte morphogenesis.

Antivascular targeting is a promising strategy for tumor therapy. This strategy overcomes many of the transport barriers and has shown efficacy in many preclinical models, but targeting epitopes on tumor vasculature can also promote accumulation in healthy tissues. We used Elastin-like Polypeptide (ELP) to form block copolymers (BCs) consisting of two separate ELP blocks seamlessly fused at the genetic level. ELPBCs self-assemble into spherical micelles at a critical micelle temperature (CMT), allowing external control over monovalent unimer and multivalent micelle forms. We hypothesized that thermal self-assembly could trigger specific binding of ligand-ELPBC to target receptors via the multivalency effect as a method to spatially restrict high-avidity interactions. We termed this approach Dynamic Affinity Modulation (DAM). The objectives of this study were to design, identify, and evaluate protein-based drug carriers that specifically bind to target receptors through static or dynamic multivalent ligand presentation.

ELPBCs were modified to include a low-affinity GRGDS or GNGRG ligand and a unique conjugation site for hydrophobic compounds. This addition did not disrupt micelle self-assembly and facilitated thermally-controlled multivalency. The ability of ligand-ELPBC to specifically interact with isolated AvB3 or CD13 was tested using an in vitro binding assay incorporating an engineered cell line. RGD-ELPBC promoted specific receptor binding in response to multivalent presentation but NGR-ELPBC did not. Enhanced binding with multivalent presentation was also observed only with constructs exhibiting CMT < body temperature. This study establishes proof-of-principle of DAM, but ELPBC requires thermal optimization for use with applied hyperthermia. Static affinity targeting of fluorescent ligand-ELPBC was then analyzed in vivo using intravital microscopy (IM), immunohistochemistry (IHC), and custom image processing algorithms. IM showed increased accumulation of NGR-ELPBC in tumor tissue relative to normal tissue while RGD-ELPBC and non-ligand ELPBC did not, and IHC verified these observations. This study shows (1) multivalent NGR presentation is suitable for static multivalent targeting of tumors and tumor vasculature, (2) multivalent RGD presentation may be suitable for DAM with thermal optimization, and (3) ELPBC micelles may selectively target proteins at the tumor margin.

As human skin ages, its ability to both repair wounds and protect them from infection declines. Several factors play major roles in this, including thinning of epidermis and loss of collagen leading to skin fragility, as well as the decline of innate immune function, though the latter has been less distinctly linked to skin aging. Given the rising aging population globally, understanding how the skin responds to injury across the life spectrum is increasingly important. This work attempts to understand specifically how innate antiviral immunity of the skin is downregulated in age. To do so, we leveraged a discovery where we determined that aging skin has a differential circadian clock, a known immunological regulator, compared to that of younger skin. The circadian rhythm is a biological clock that uses a transcriptional-translational feedback loop to set up patterns of activity throughout the body. This loop uses positive transcription factors BMAL1 and CLOCK, which set up their own repressors, including the PER and CRY family of proteins. This rhythm also influences biological functions throughout the body. In the scope of this work, we became interested in the fact that circadian rhythms were found to influence epithelial repair in injury, antiviral immunity and interferon stimulated genes. To begin our study, we asked whether the known repair and immune dysfunctions of aging skin could be possibly tied to a dysfunctional circadian rhythm. Using qRT-PCR, we found that aging murine skin has a decreased circadian transcription when compared to that of younger skin. Such a phenotype was replicated by human keratinocyte studies using serial passaging as an aging surrogate. Having determined that aging does indeed play a role in regulating cutaneous circadian rhythms, we set out to determine what immune mechanisms of the skin are regulated by this aging-circadian axis. Specifically, we tested circadian regulation of antiviral proteins. Antiviral proteins of distinct families and functions all protect the skin from pathogen invasion. Prior work by our lab had shown that antiviral proteins were induced by skin wounding in a pathway dependent on the cytokine IL-27. Using approaches including qRT-PCR, flow cytometry, and immunofluorescence, we determined that aging skin wounds not only have an attenuated antiviral protein response, but also contained reduced numbers of CD301b+ immune cells that produce IL-27. These distinct immune deficiencies lead to an unprotected skin wound microenvironment in aging skin. However, little is understood about the molecular mechanisms responsible for the antiviral immune deficiencies in the aging skin. To address this, we began by probing publicly available datasets, where we found that the expression of antiviral proteins had 24-hour rhythms of expression in murine skin. Similarly, we found that that rhythmic expression of antiviral proteins occurs in human keratinocytes that were synchronized in a circadian fashion. Additional support for a direct line of circadian regulation of antiviral proteins came from circadian siRNA studies, where we knocked down expression of circadian gene CLOCK and saw an associated downregulation in antiviral proteins within human keratinocytes. Further, we demonstrate via qRT-PCR that murine skin harvested at different time points have different antiviral protein mRNA levels. Subsequent computational analysis showed that Bmal1-/- murine skin is deficient in antiviral protein expression, establishing a direct link between circadian factors and antiviral proteins. In order to better understand the effect of circadian rhythms on wound immune responses, we made use of a number of experimental models, including both Bmal1-/- and ClockΔ19 mutant mice, as well as wild type animals. We wounded wild type animals at distinct time-of-day, and found that the level of antiviral proteins display time-of-day responses, peaking at 8pm. Using circadian mutant mice, we found that these animals have attenuated wound responses with respect to antiviral protein induction; specifically, wounded ClockΔ19 mice do not produce antiviral proteins to the same extent as wild type mice. We were able to tie this directly to IL-27 signaling in two distinct manners. Firstly, using flow cytometry, we found that the CD301b+ cells that produce IL-27 in response to wounding are reduced in number in circadian mutant mouse skin, and moreover, produce less IL-27 as measured by median fluorescence intensity. To determine the role of IL-27 in the time-of-day response of wound-induced antiviral protein expression, we wounded IL-27fl/fl-LysM-Cre mice at two distinct times-of-day and measured antiviral protein production. These mice lack IL-27 production from myeloid cell lineages, including CD301b+ cells. We found that loss of IL-27 diminished time-of-day differential expression of antiviral proteins in wounds, further suggesting that the link between circadian rhythms and antiviral proteins was in fact in part mediated by IL-27. To further our understanding of the cytokine milieu of circadian wounds, we also wounded mice that lacked Type I interferon receptor (IFNAR1) expression and found that loss of Type I interferon signaling also blunted time-of-day antiviral protein responses. These data support a role of both interferons and IL-27 in circadian antiviral protein induction. In order to provide a functional aspect to these findings, we infected wildtype and circadian disrupted keratinocytes and human skin with Herpes Simplex Virus Type I (HSV1). We measured HSV expression in the skin by both immunofluorescence and visual characterization as well by qPCR for viral component UL29. We found that circadian disruption of either BMAL1 or CLOCK sensitizes keratinocytes to HSV1 infection in vitro. On the other side of this spectrum, we questioned whether circadian enhancing drugs, including the compounds SR8278 and nobiletin, can activate circadian rhythms in skin cells and improve skin defense against HSV1 infection. Using a BMAL1:Luciferase reporter, we characterized both drugs as having a circadian augmenting effect in keratinocytes. Upon infection with HSV1, both SR8278 and nobiletin protected human skin from viral spread. Further, we found that SR8278’s antiviral effect was predicated on circadian activity, as BMAL1 and CLOCK siRNA knockdown in keratinocytes lessened the drugs effect. To determine mechanism of circadian drug’s antiviral activity, we tested whether our circadian drugs activate canonical antiviral signaling pathways, such as OAS and IFITM. We found that via qPCR, circadian drugs require the presence of these proteins to fight virus effectively. As a study of clinical relevance, we evaluated the role of acyclovir treatments alongside our circadian drugs. We found that circadian drugs SR8278 and nobiletin did not synergize their effects with acyclovir at a variety of doses tested. We found that acyclovir, as expected, broadly suppressed HSV1 activity at even low doses in keratinocytes, an effect that circadian augmentation was unable to potentiate. This could be due to a number of factors, including dosage optimization and viral susceptibility to drug. However, given the rise of acyclovir resistant HSV, our novel approach may be clinically viable. In particular, we believe this may be a viable treatment platform for aging skin infections; to this end, we tested the ability of SR8278 to suppress HSV1 infection in the skin of mice over a year of age. SR8278 significantly reduced viral spread in this model, suggesting that circadian augmentation may be a useful clinical adjunct in aging skin infections. To determine if these findings were applicable to other non-herpes family viruses that infect the skin, we turned to a model of West Nile Virus infection. West Nile Virus is a mosquito borne illness with increasing range and infection number in the United States. Moreover, it is inoculated through the skin before causing neurological infection, a pathway similar to herpes viruses. Also similar to herpes viruses, West Nile virus is a far more pressing clinical issue in aging populations, who fare worse with this viral infection. Most importantly, there are no currently specific treatments for West Nile Virus infections. Using our HSV infection data as a starting point, we found that circadian drug treatments suppressed West Nile Virus levels in infected keratinocytes. Other work conducted over the course of this PhD encompassed aspects of both circadian and IL-27 signaling in the skin. Using human keratinocytes, we endeavored to understand what environmental factors could drive altered circadian rhythms in the skin, for either elderly or younger tissue. While dogmatically, circadian rhythms are patterned from the brain to the whole body, we built on recent work showing a light dependent murine cutaneous clock by showing that mock sunlight can alter circadian expression in human keratinocytes, without other stimuli present. Further study is needed to understand how our skin’s clock responds to sun mechanistically. Overall, my work over the course of this PhD has established a link between aging, circadian rhythms, and antiviral immunity, and underpinned the important role of the cytokine IL-27 and type I interferon on cutaneous wound responses to a variety of pathogens. This work will provide possible therapeutic avenues, particularly for aging skin, in how to address skin wound care in safe, biologically relevant ways via circadian rhythm exploitation.

Normal cells require input from extrinsic growth factors to control proliferation and survival. Recent studies have demonstrated that these same extrinsic signals also regulate cellular metabolism to ensure that metabolism adequately supports the demands of cell function, proliferation, and cell survival. The PI3K/Akt pathway is downstream of many growth factors and can promote both glucose metabolism and cell survival. Aberrant activation of the PI3K/Akt pathway is common in cancer, and its activation can contribute to the growth factor independence that is a hallmark of neoplastic cells. Metabolic demand is high in stimulated and leukemic T cells, and activation of Akt can increase glucose metabolism to meet these requirements. There is great interest in targeting the unique metabolism of cancer cells for cancer therapy, thus making an understanding of the interaction of metabolism and cell death essential.

Akt is also anti-apoptotic and can inhibit cell death by regulating members of the Bcl-2 family. Interestingly, the ability of Akt to prevent cell death is inextricably linked to its metabolic function. Several recent studies have demonstrated that glucose metabolism can affect Bcl-2 to family members to promote cell survival, but the role of Akt-dependent glucose metabolism in the regulation of Bcl-2 family members is not understood. Using a model of growth factor withdrawal-induced apoptosis, we show that Akt prevents cell death by maintaining glucose metabolism to regulate the Bcl-2 family members Puma and Mcl-1, and demonstrate the importance of this pathway in the survival of stimulated T lymphocytes and leukemia.

After growth factor withdrawal, active Akt suppressed Puma induction in abundant glucose, but Puma was rapidly upregulated in glucose-deficient conditions and was necessary and sufficient to promote efficient cell death. Importantly, glucose was not uniquely required, as provision of alternative mitochondrial fuels allowed Akt to suppress Puma and maintain survival. This metabolic regulation of Puma was mediated through partially p53-dependent transcriptional induction as well as control of Puma protein stability.

In addition to inhibiting Puma expression, active Akt prevented the loss of Mcl-1 after growth factor withdrawal by sustaining Mcl-1 protein synthesis in a glucose-dependent manner. Mcl-1 was essential for preventing Bim-induced apoptosis, as Akt could not inhibit Bim induction after growth factor deprivation. Slowing of Mcl-1 synthesis by inhibiting glucose metabolism reversed Mcl-1-mediated resistance of leukemic cells to the Bcl-2 inhibitor ABT-737. Importantly, Akt and glucose-reliant Mcl-1 expression required mTOR-dependent phosphorylation of 4EBP, and treatment with mTOR inhibitors also reversed ABT-737 resistance.

Together, this study demonstrates that Akt promotes cell survival by preventing metabolic checkpoints that stimulate Puma expression and stability and inhibit Mcl-1 synthesis, advancing our understanding of the links between metabolism and cell death. These studies highlight the importance of cellular metabolism--including a potential role for the alternative sugar fructose--in cancer cell survival that may provide a mechanistic understanding to drive development of metabolism-targeted cancer therapies.

Pseudomonas aeruginosa, known as one of the leading causes of morbidity and mortality in cystic fibrosis (CF) patients, secretes a variety of virulence-associated proteases. These enzymes have been shown to contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds and chronic wounds. Our lab has previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is also highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known for their contribution to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. With this in mind, we hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm microcolony composition to increase cellular density, while decreasing matrix polysaccharide content and resistance to the antibiotic colistin. We recreate this phenotype using a pellicle biofilm model, in which bacteria are grown statically at the culture air-liquid interface, demonstrating that these phenotypes are not dependent on the coculture host cell substrate. We additionally show that OMVs from PaAP expressing strains, but not PaAP alone or in combination with PaAP deletion strain-derived OMVs, could complement this phenotype. Finally, we found that OMVs from PaAP-expressing strains cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. OMVs mediated the detachment of biofilms formed by both non-self P. aeruginosa strains and K. pneumoniae, another respiratory pathogen, showing that this process may also be relevant in polymicrobial communities and acts on non-P. aeruginosa derived substrates. Our findings represent novel roles for OMVs and the PaAP aminopeptidase in the modulation of bacterial biofilm architecture.

Our group has pioneered the development of a live-attenuated poliovirus, called PVSRIPO, for the purpose of targeting cancer. Despite clinical progress, the cancer selective cytotoxicity and immunotherapeutic potential of PVSRIPO has not yet been mechanistically dissected. Defining such mechanisms may inform its clinical application.

Herein I describe the discovery of a mechanism by which the MAP-Kinase Interacting Kinases (MNKs) regulate PVSRIPO cytotoxicity in cancer. In doing so, I delineate a novel, intricate network connecting the MNK and mTOR signaling pathway that regulates activity of a splicing kinase called the Ser-Arg Rich Protein Kinase (SRPK), and define SRPK as an impediment to IRES mediated translation. Moreover, I demonstrate that MNK regulates mTORC1 associations that determine its substrate proximity and thus, activity. In a collaborative effort, we found that PVSRIPO oncolysis produces antigen specific, cytolytic anti-tumor immunity in an in vitro human system and that much of the observed adjuvancy is due to the direct infection of dendritic cells (DCs) by the virus itself; implicating PVSRIPO as a potent adjuvant. In summary, oncogenic signaling in part through MNK leads to cancer specific cytotoxicity by PVSRIPO that engages an inflammatory environment conducive to DC activation and antigen specific T cell antigen immunity.

The APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) family of cytidine deaminases is one of the most common endogenous sources of single base substitution mutations in human cancer. Accordingly, APOBEC enzymes represent a major source of intratumor genetic heterogeneity and have been associated with immunotherapy response in diverse cancer types. However, the consequences of APOBEC mutagenesis on tumor progression in vivo are not well understood. To address this, I developed several murine tumor models with inducible APOBEC3B expression and studied the contribution of APOBEC activity to tumor evolution and immunogenicity. First, I explored the effects of APOBEC activity on tumor relapse using a murine model of mammary tumor recurrence. APOBEC activity led to a significant acceleration in tumor recurrence following the strong selective pressure of oncogenic driver signaling loss. Recurrent APOBEC tumors had undifferentiated histological features and large, irregularly shaped nuclei containing defects like micronuclei, multinucleation, and chromatin bridges. I found that recurrent APOBEC tumors amplified the therapy resistance-associated oncogene, c-Met, on circular extrachromosomal DNA, likely driving the proliferation of the recurrent cancer cells. Second, because APOBEC mutational signatures are enriched in the majority of HER2-positive breast cancer patients, I used a syngeneic HER2-driven mammary tumor model to study the effects of APOBEC activity on the tumor immune microenvironment. I found that APOBEC activity induced an antitumor adaptive immune response and CD4+ T cell-mediated tumor growth inhibition. While polyclonal APOBEC tumors had a moderate growth defect, clonal APOBEC tumors were almost completely rejected by the immune system, suggesting that APOBEC-mediated genetic heterogeneity limits the antitumor adaptive immune response. In human breast cancers, the relationship between APOBEC mutagenesis and immunogenicity varied by breast cancer subtype and the frequency of subclonal mutations. Consistent with the observed immune infiltration in murine APOBEC tumors, APOBEC activity sensitized HER2-driven breast tumors to checkpoint inhibition. This work provides a mechanistic basis for the sensitivity of APOBEC tumors to checkpoint inhibitors and suggests a rationale for using APOBEC mutational signatures and clonality as biomarkers predicting immunotherapy response in HER2-positive breast cancers. In conclusion, I’ve identified a novel role for APOBEC activity in generating chromosomal instability, consisting of mitotic errors, oncogene amplification, and extrachromosomal DNA formation to promote tumor recurrence. Moreover, APOBEC activity also stimulated an antitumor adaptive immune response and sensitized tumors to immunotherapy.

Phase separation of macromolecules is a critical phenomenon for the human condition. This phenomenon has also been exploited for biotechnological development to improve human morbidity and mortality. However, there is still much more to learn regarding how this behavior is encoded within a protein sequence. Thus, this thesis seeks to 1) further explore the sequence space to understand how phase separation is encoded, with an emphasis on polypeptides with upper critical solution temperature (UCST) transitions and 2) use this phase separation to control availability of macromolecules at various length scales.

Using traditional molecular biology techniques, we will recombinantly express and purify a large number of polypeptides with variable sequence composition and sequence architecture. Then, using traditional polymer science and material science techniques combined with microscopic techniques that span the macro-scale and nano-scale, we will characterize their phase separation behavior and the interaction of these materials with biological systems.

We developed a practical mutation strategy that allows for complete control of the UCST binodal line in physiologic conditions that is useful for de novo design of artificial IDPs with UCST phase behavior. We evaluated the interaction of these polypeptides and their phase separation in the presence of bacterial, eukaryotic cells and in mice demonstrating how this binodal line fused to biological active partners can control biologic functions.

In bacteria, we made artificial phase separated puncta, akin to naturally occurring phase separated droplets, that have non-canonical function, demonstrating how primary features of the polypeptide chain affect enzymatic function. We created block co-polypeptides comprised of UCST and LCST protein sequences that exhibit remarkably tunable and robust nanoscale self-assembly into spherical micelles, worm-like micelles and vesicular structures capable of displaying large targeting domains on their surface. In the presence of eukaryotic cells, these nanomaterials can dramatically increase polypeptide uptake, increasing the avidity of the targeting molecule by over 1000-fold.

Finally, we demonstrated that phase separated polypeptides can sequester an active peptide GLP-1 from systemic circulation, controlling the peptide’s bioactivity through control of the phase diagram. Taken together, we demonstrate the universal power of the phase diagram, across many length scales, where the transducing agent for controlling biological activity is an engineered, repetitive polypeptide sequence.