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Delineating the Mechanisms of Misfolded Endoplasmic Reticulum (ER) Luminal Protein Retrotranslocation for ER-Associated Degradation
Get PDFSecreted, plasma membrane, and resident proteins of the secretory pathway are synthesized in the endoplasmic reticulum (ER) where they undergo post-translational modifications, oxidative folding, and subunit assembly in tightly monitored processes. An ER quality control (ERQC) system oversees protein maturation and ensures that only those reaching their native state will continue trafficking into the secretory pathway to reach their final destinations. Proteins that fail quality control must be recognized and eliminated to maintain ER proteostasis. The ER-associated degradation (ERAD) was discovered nearly 30 years ago and entails the identification of improperly matured secretory pathway proteins and their retrotranslocation to the cytosol for degradation by the ubiquitin-proteasome system. While multiple ER-Associated Degradation (ERAD) components have been identified and their roles elucidated, it remains less clear how folded domains on ERAD clients complicate their extraction from the ER and degradation. To address this, we used several luminal ERAD substrates with well-defined structural properties. Deglycosylation and digitonin permeabilization assays were used to monitor client extraction from the ER. Our fully unfolded clients were released into the cytosol when the proteasome was inhibited. Conversely, ERAD clients possessing a single folded domain were completely retrotranslocated but were not released from the ER membrane without proteasome function. These clients were fully reduced, but still retained structure in their folded domain. Overall, our data argue that ubiquitinated clients with well-folded domains can be dislocated from the ER in a p97-dependent manner, but proteasome activity is required to fully release them from the cytosolic side of the ER membrane and the ERAD extraction machinery.To decipher key steps in the extraction of non-glycosylated ER luminal proteins, and to determine how Hrd1, p97, and the proteasome contribute to the retrotranslocation of clients, we developed a novel in-cell biotin-based reporter system. The bacterial BirA biotin ligase was tethered to FAM8A1, a component of the retrotranslocon that interacts with Hrd1, thus positioning the biotin ligase near the cytosolic exit site. The non-secreted immunoglobulin (Ig) κ light chain (NS1 κ LC), which is composed of a poorly folded N-terminal domain and a well-folded C-terminal domain was used in the biotinylation assays. NS1 was modified at either the N- (BAP-NS1) or C- (NS1-BAP) terminus with a biotin acceptor peptide (BAP) allowing us to detect cytosolic exposure of both ends of this ERAD client. We established that BAP-tagged NS1 constructs were still ERAD substrates and that BirA-tagged FAM8A1 still assembled with the retrotranslocon. We found that both termini of NS1 were readily biotinylated when the proteasome was inhibited by MG132 treatment, or when dominant negative constructs of Hrd1 and p97 were expressed. To differentiate between full extraction of NS1 and partial exposure of the termini to the cytosolic side of the ER, we permeabilize the plasma membrane with digitonin. Cytosolic proteins were released, but both biotinylated and non-biotinylated NS1 remained cell-associated. This argues that the biotinylated light chain was not fully extracted from the ER when NS1 degradation was inhibited with either MG132 or with the Hrd1 and p97 mutants. The digitonin permeabilized cells expressing BAP-NS1 were treated with proteinase K to assess how much of this client was exposed to the cytosolic side of the ER. We found that the NS1 κ light chain was only partially extracted when cytosolic ERAD components (proteasome and p97) were impaired. These data indicate that the termini of this ERAD client can be inserted into the retrotranslocon and “sample” the cytosolic side but require the action of an E3 ligase, the p97 complex and the proteasome to be fully extracted
An Update on Pharmaceutical Strategies for Oral Delivery of Therapeutic Peptides and Proteins
Get PDFPeptides and proteins are imperative for the human body and play crucial roles in governing various bio-chemical processes. Recent advances in molecular biology and biochemistry helped in understanding the role of these endogenous macromolecules in different pathological and disease conditions. Currently, small molecule drugs (\u3c 900dalton) in comparison to the therapeutic peptides and proteins-based drugs (TPP) dominate pharmaceutical market. However, the game is changing with the recent advances of biotechnological tools like recombinant DNA technology, solid phase protein synthesis etc., which enabled large-scale production of therapeutic peptides and proteins. The Success of Human Insulin, the first FDA approved commercial recombinant protein based therapeutic in 1982, revolutionized the field of TPPs. The number of FDA approved TPPs reached about to ~239 in 2017 compared to where it was only ~130 in 2008. Rapid progress in this sector can be attributed to several advantages of proteins and peptides over small molecule drugs both financially and clinically. From a clinical perspective, proteins and peptides are inherently more specific to the target site than the small molecules drugs, which lead to less interferences with normal biological system of the patient and caused minimal off-target side effects. A handful of proteins which are used for different clinical complications are less immunogenic because they are produced in the body naturally. Furthermore, proteins and peptides also take part in several complex and complicated biological processes, which is difficult to be to be mimicked by the small molecule drugs. From a financial standpoint, median total pre-market development times were shorter for biologics (10.6 years) than the small molecules drugs (12.6 years) estimated using Merck Index. In 2009, US Congress passed the Biologics Price Competition and Innovation Act (BPCIA) which gave new biologics 12 years of guaranteed exclusivity. The most commonly utilized routes for administering TPPs are I.V, I.P or I.M injections, which largely suffer from patient compliances. There are ~350 TPPs under clinical development and among them only 2 are given orally which is Interferon-α and Human growth hormone. Currently, most efforts in both industry and academia are centered around enhancing bioavailability of orally administered TPPs which typically are less than 1%. Oral administration is the non-invasive, most preferred route of drug administration for the patients. Furthermore, oral dosage forms are cheaper to manufacture as well as to administer, because they do not need to be produced under sterile conditions or administered in clinics. However, unfavorable physicochemical characteristics of TPPs like high molecular weight, hydrophilicity, poor stability in the physiological conditions, short biological half-life, low permeability through the epithelial barrier in the small intestine put up a massive barrier in the development of orally available dosage forms of TPPs. In this review, we will discuss the challenges associated with oral delivery of TPPs and the ongoing efforts to solve them
The Role of Exosomal Transport of Viral Agents in Persistent HIV Pathogenesis
Get PDFHuman immunodeficiency virus (HIV) infection, despite great advances in antiretroviral therapy (ART), remains a lifelong affliction. Though current treatment regimens can effectively suppress viral load to undetectable levels and preserve healthy immune function, they cannot fully alleviate all symptoms caused by the presence of the virus, such as HIV-associated neurocognitive disorders (HAND). Exosomes are small vesicles that transport cellular proteins, RNA, and small molecules between cells as a mechanism of intercellular communication. Recent research has shown that HIV proteins and RNA can be packaged into exosomes and transported between cells, to pathogenic effect. This review summarizes the current knowledge on the diverse mechanisms involved in the sorting of viral elements into exosomes and the damage those exosomal agents can inflict. In addition, potential therapeutic options to counteract exosomemediated HIV pathogenesis are reviewed and considered
Roles of Cytosolic Nucleic Acid Sensors in Cancer and Infection
Get PDFPattern recognition receptors are innate immune sensors that recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) with crucial roles in host defense against microbial infection, autoimmune diseases and cancer. Cytosolic nucleic acids including DNA and RNA originate from pathogens or self-cells, which form major groups of PAMPs and DAMPs. A range of nucleic acid sensors have evolved to sense various types of nucleic acids. How different DNA-sensing pathways regulate microbial infection and cancer is the focus of this dissertation.
Stimulator of IFN genes (STING) is a cytosolic innate immune sensor for cyclic dinucleotides that also serves a dual role as an adaptor molecule for a number of intracellular DNA receptors. A physiological role for STING in cancer was previously unknown. We showed that STING-deficient mice were highly susceptible to colitis-associated colorectal cancer. Colons of STING-deficient mice exhibited significant intestinal damage and overt proliferation with increased levels of pro-inflammatory cytokines during early stages of tumorigenesis, uncovering an unexpected and important role for STING in mediating protection against colorectal tumorigenesis.
Absent in melanoma 2 (AIM2) forms an inflammasome with ASC and caspase-1 upon recognition of double-stranded DNA (dsDNA) in the cytosol leading to caspase-1 activation and caspase-1-dependent pyroptosis and release of cytokines IL-1 and IL-18. Mutations in AIM2 are frequently identified in patients with colorectal cancer, but how AIM2 modulates colonic tumorigenesis is unknown. We found that AIM2-deficient mice were hypersusceptible to colonic tumor development. While production of inflammasome-associated cytokines and other inflammatory mediators was largely intact in AIM2-deficient mice, intestinal stem cells lacking AIM2 were prone to uncontrolled proliferation. Aberrant Wnt signaling expanded a population of tumor-initiating stem cells in the absence of AIM2 driving the tumor development in AIM2-deficient mice. In addition to its role in cancer, AIM2 recognizes bacteria including Francisella tularensis subspecies novicida (F. novicida) and induces inflammasome responses. Type I interferon (IFN) signaling drives activation of AIM2 inflammasome in F. novicida-infected macrophages; however, the relative contribution of IFNs and inflammasome responses in host defense against F. novicida infection is less understood. We found intact AIM2 inflammasome responses in mice lacking type I IFN signaling during infection with F. novicida. Lack of type I IFN signaling conferred protection to F. novicida infection in contrast to the increased susceptibility in AIM2-deficient mice. Interestingly, mice lacking both AIM2 and IFNAR2 were protected against the infection indicating a dominant role for type I IFNs in mediating detrimental responses despite the protective AIM2 inflammasome responses. Gasdermin D (GSDMD) is activated by caspase-1 to generate pores on the plasma membrane to induce pyroptosis downstream of the AIM2 inflammasome. We also demonstrated that mice lacking GSDMD were highly susceptible to F. novicida infection. Interestingly, GSDMD is required for optimal caspase-1 activation during F. novicida infection, providing protection to the host during the infection.
In addition, we identified differential mechanisms regulating expression of inflammasome-associated cytokines IL-1β and IL-18. IL-1β is only induced in response to inflammatory stimuli and its expression is not sustained during chronic treatment, while IL-18 is constitutively expressed and further induced after the stimulation in a type I IFN signaling-dependent manner.
Overall, this dissertation addresses protective roles for cytosolic nucleic acid-sensing molecules, STING and AIM2, in colon cancer, uncovers an interplay between the AIM2 inflammasome and type IFN signaling during F. novicida infection, and demonstrates a novel function for GSDMD in regulating AIM2 inflammasome. The dissertation finally describes distinctive mechanisms governing inflammasome-associated cytokines IL-1β and IL-18
The Tumor Microenvironment Regulates Retinoblastoma Cell Survival
Get PDFRetinoblastoma (Rb) is the most common intraocular malignancy in children comprising 4% of all pediatric tumors. Early intervention increases survival rates up to 95% in developed countries; being survival rates associated to socio-economic status. Despite the high survival rates in developed countries, preservation of the eye and vision are continuing challenges in the management of Rb. Vitreous seeds constitute the greatest challenge in treatment of Rb. The unique location of these seeds within the vitreous makes them difficult to treat. Viable seeds showed reduced proliferative capacity and metabolism. making the majority refractory to current chemotherapeutics. This prompted the development of new delivery routes for chemotherapeutics, such as intra-ophthalmic artery, intravitreal, and subconjunctival administration. Still ocular salvage rates have not exceeded 70% in over two decades. Novel, safer therapies are required but there is a fundamental lack of knowledge about the biology of the tumor for the development of targeted therapies; the vitreous, as it surrounds the seeds; and the interactions between the vitreous and the seeds.
Despite the current understanding of how cancer is a multifaceted disease full of complex cellular and protein interactions (commonly referred to as the tumor microenvironment, TME), the immunology of the vitreous microenvironment and the role it plays in the sustainment of seeds in Rb disease is poorly understood. In this study, we begin investigating Rb tumor cells and how they alter the TME by examination of matrix metalloproteinases, a family of enzymes involved in degradation of the extracellular matrix and tissue remodeling heavily implicated in tumor migration and survival. We identified two gelatinases, MMP-2 and MMP-9, to be expressed in Rb cell lines and demonstrated by pharmacological inhibition and genetic knockdown, a role for these gelatinases in Rb cell migration, invasion, and survival. Additionally, we demonstrated how secretion of VEGF, involved in angiogenesis, and TGFβ, involved in metastasis, were altered by MMP-2 and MMP-9 pharmacological inhibition.
As MMPs are pivotal for the tumor and extracellular matrix interactions within the TME leading to tissue invasion, we then transitioned to the vitreous, investigating the vitreous as a TME and how it sustains Rb. Multiple proteins are found within the soluble phase of the vitreous that are associated with ocular pathological processes, including diabetes retinopathy and proliferative vitreoretinopathy. Therefore, we compared the presence of a small cohort of proteins associated to ocular pathologies, to healthy vitreous and to Rb patients’ vitreous samples, identifying high expression of Platelet-Derived Growth Factor Receptor β (PDGFRβ) and its ligand PDGFBB in the Rb samples. Additional studies of ex vivo healthy human vitreous, murine Rb xenografts, and patient-derived Rb xenograft tissues, measured high activity of the PDGF-PDGFRβ signaling pathway in diseased, but not healthy tissue. We focused the next part of this work on the role PDGFRβ plays in vitreous seeds. To investigate this pathway in depth we used established Rb cell lines, namely Y79-the metastatic and aggressive model, and Weri-1 Rb, the non-metastatic model. Our work demonstrated PDGFRβ was overexpressed in Y79 cells, the metastatic model of Rb, in vitro. To provide mechanistic insight, we utilized the tyrosine kinase inhibitor imatinib mesylate (IM), and demonstrated PDGF-PDGFRβ signaling pathway regulates Rb cell proliferation, invasion, and survival. We found increased PDGF-PDGFRβ signaling resulted in higher activity of the p53-inactivator, MDM2, as well as the pro-inflammatory NFβB pathway, both of which are involved in tumor survival. As there is a lack of models for vitreous seeding, we utilized magnetic nanoparticles to generate the first in vitro vitreous seed model and demonstrated similar features between an ex vivo seed and one of our in vitro magnet-generated 3D tumor spheroids, validating our system. Using this novel technology, we recapitulated our in vitro 2D work and demonstrated that inhibition of the PDGF-PDGFRβ signaling pathway results in decreased spheroid size. PDGFRB gene knockdown by siRNA confirmed the results with the pharmacological agent showing these results were PDGF-PDGFRβ signaling-specific.
Next, we tested the effects of IM, as a potential Rb therapy, in retinal endothelial cells h(RECs) as retinal endothelial cell-associated toxicities are one of the challenges with conventional chemotherapies in Rb. We measured the capacity of hREC to proliferate and for tube formation in the presence of the therapy. Our results demonstrated neither proliferation nor tube formation of hRECs changed when exposed to IM. Upon further examination, we demonstrated the absence of PDGFRB mRNA expression in hRECs. Taken together these results illustrate the potential use of anti-PDGFRβ therapy as a targeted therapy in Rb.
The last section of the study investigated the expression and function of members of the ATP-binding cassette (ABC) transporters as well as the multifunctional glycoprotein CD44 in Rb, as one of the properties of vitreous seeds is their resistance to chemotherapy. We discovered heterogeneous expression of multiple ABC transporters in vitro using Y79 Rb cell line and ex vivo using Rb vitreous samples. In addition to expression, the transporters efflux activity was determined. CD44 mRNA, whose protein is considered a promoter of chemoresistance, was highly expressed in naïve vitreous from Rb patients. These results illustrate that chemoresistance is not induced by treatment and is, instead, an innate feature of vitreous seeds. Flow cytometry analyses established (1) a high correlation between active, or phosphorylated PDGFRβ (p-PDGFRβ) and CD44; and (2) inhibition of p-PDGFRβ resulted in decreased CD44, as well.
Collectively, this study demonstrated the Rb TME plays a crucial role in Rb tumor properties. MMP-2 and MMP-9 are highly expressed in Rb and allow degradation of the ECM in tissue, increasing migration and invasion of Rb. Additional work focused on vitreous seeds and identified increasing signaling activity of the PDGF-PDGFRβ signaling pathway in multiple Rb models, including in vitro cell lines, ex vivo Rb patient vitreous samples and tissue, as well as in a murine xenograft system. Next, we identified this pathway as a potential target, as pharmacological and genomic inhibition of the PDGF-PDGFRβ signaling pathway by IM and PDGFRB siRNA resulted in decreased proliferation, invasion, resistance, and survival. These cellular functions appear to be mediated by the downstream targets MDM2 and NFβB. Our initial studies demonstrated a lack of drug-associated toxicity on hRECs with IM. This work is a further step in our quest for targeting vitreous seeding
Keeping Up with Trends in Your Library: Simple and Speedy Ways to Assess Users’ Needs
Get PDFObjective: To find simple, fast, and easy ways to assess library users’ needs and to get real-time responses in order to shorten the interval between deploying surveys, gathering responses and implementation of solutions. In the past the library employed email surveys to assess user needs but the process was inefficient and ineffective due to low response rates and time spent creating online surveys. Methods: The library team began using paper “micro-assessments” to understand users’ needs. Short paper surveys of no more than five questions, were deployed at three different times throughout the academic year: (1) Medical students were handed surveys during one of the library’s weekly events about their use of library-issued iPads; (2) Surveys were left on each study space in the library quiet room over the course of one week asking about students’ opinions on food being allowed in the library quiet room; (3) Faculty were handed surveys during departmental meetings to assess their interest in scholarly publishing topics. Completed surveys were collected and results were tabulated the same day. Results: Results of the student iPad micro-assessment helped librarians understand students’ use of library-issued iPads. Feedback from the second student survey informed policy decisions in regards to allowing food in the library quiet room. Finally, results of the faculty micro-assessments led to the development of short presentations on scholarly communication topics, delivered by librarians during faculty departmental meetings, as well as one-on-one EndNote training sessions and a Getting Published Workshop
Leading the Way: Establishing a versatile EBSCO e-audiobooks leadership collection tosupport talent development initiatives and employee growth in a large healthcare system
Get PDFUsing a Reflexive Process to Investigate Organizational Change: The Use of the Research Spider Matrix
Get PDFThe primary objective was to assess the research competencies (knowledge, experience, and skills) of librarians at an academic health sciences library using the Research Spider matrix (Smith et al., 2002). This was motivated by the shift from a traditional reference model to a research-centric paradigm
