Sifers helped pioneer the initial mechanistic analysis of the biological systems that manage glycoprotein homeostasis (i.e. glycoproteostasis) in the mammalian secretory pathway. Using alpha1-antitrypsin deficiency as a medically relevant paradigm and client, his lab characterized the processes of chaperone-assisted glycoprotein folding as a means of conformation-based intracellular retention and proposed and characterized the mannose timer hypothesis as an initial step in N-glycan-targeted proteolysis (quality control). Subsequently, his team identified the underlying contribution of the unfolded protein response (UPR) and elucidated how compromised quality control can function as an etiologic agent of infantile liver cirrhosis.
Statement of Problem: Although inherited information exists within a genetic material, defects in the encoded proteins are responsible for manifestations associated with abnormal biology. Therefore, a need exists to mechanistically define the proteostasis systems responsible for managing the cellular proteome as a means to eventually identify novel therapeutic sites for disease intervention. To this end, we have monitored the fates of newly synthesized proteins that are translocated into the secretory pathway, many of which are subjected to asparagine(N)-linked glycosylation. In addition to facilitating proper protein folding, modification of the appendage flags misfolded N-glycoproteins for elimination by ???ER-associated Degradation??? (ERAD). The currently accepted flagging mechanism involves the opportunistic cleavage of alpha-1,2-mannose units. Although this crucial event was initially thought to involve ER mannosidase I (Man1b1), recent evidence indicates that the protein is not a component of the mammalian glycoprotein quality control interactome, localizes to post-ER compartments, and does not require enzymatic activity to promote N-glycoprotein degradation. Methodology & Theoretical Orientation: The present study sought to define the contribution of Man1b1 to the operation of an apparently ???unconventional??? ERAD client recruitment system. The effects of wildtype and selectively mutated forms of recombinant human Man1b1 on the fates of selected ERAD clients were monitored through the use of pharmacologic inhibitors, metabolic radiolabeling, immunoprecipitation, and western blotting. Findings: Distinct populations of Man1b1 have identified that exhibit different intracellular trafficking patterns, unique functional partners, and unique client specificities. Conclusion & Significance: An unexpected level of functional plasticity exists in the proteostasis network of the secretory pathway, extending the role of a specific mannosidase beyond that of limits of Glycobiology.
Myron R Szewczuk is Full Professor of Immunology and Medicine, Queen’s University, Kingston, Ontario Canada for the past 37 years. Dr Szewczuk’s recent research has focused on the role of glycosylation in receptor activation with a particular focus of Toll-like, nerve growth factor Trk, eGFR and insulin receptors. He has discovered a novel receptor-signaling platform and its targeted translation in multistage tumorigenesis.
Neuraminidase-1 (Neu1) has recently emerged as a central target in the sialidase-mediated regulation of tumorigenesis. Recent evidence indicates that Neu1 plays a much more profound role in human cancers than previously expected. In clinical setting its being essential that targeted therapies are to circumvent multistage tumorigenesis, which includes genetic mutations at the different growth factor receptors, tumor neovascularization, chemoresistance, immune-mediated tumorigenesis and the development of tissue invasion and metastasis. Firstly, the cell-surface molecular signaling platform will be described as controlling Neu1 sialidase activity, and discuss its relevance in cancer cell signaling. Second, the current understanding of Neu1 activity associated with cancer development will be summarized, and outline the key roles of Neu1 during various stages of tumorigenesis, including regulation of growth factor receptor signaling, control of Toll-like receptor (TLR) signaling and immune-mediated tumorigenesis, regulation of epithelial-mesenchymal transition (EMT), metastasis and acquired chemoresistance, and regulation of tumor vascularization.
Professor Jose Kovensky has PhD from the Universidad de Buenos Aires (Argentina, 1992). He did his postdoctoral research at the Ecole Normale Superieure (Paris, France, 1994-1995). After being Professor of Organic Chemistry in Argentina, he got a Full Professor position in Amiens in 2002. He has been the principal investigator of several projects financed by the Regional Council or Picardie Region, binational projects France-Germany, France-Argentine, and partner in European Projects. He has directed or co-directed 12 PhD theses. He is a co-author of more than 80 publications (articles, book chapters, patents). He has a wide experience in the synthesis and modification of oligosaccharides, in particular, uronic acid containing oligosaccharides, sulfonated oligosaccharides, glycosaminoglycans, multivalent glycoclusters, and sugar-based surfactants.
Carbohydrates play an essential role in several biological functions associated with selective interactions with cellular proteins that govern a wide variety of life processes. In living organisms, these high-affinity carbohydrate-protein interactions occur through multivalent contacts, characterized by an enhancement of the affinity when the sugar ligand is presented in a cluster rather than alone. Several biomimetic approaches have been developed in order to prepare synthetic multivalent neoglycoconjugates to interfere with a series of pathological events such as infections due to viruses and bacteria, tumor progression and migration, and inflammation processes. We constructed multivalent glycoconjugates on oligosaccharide scaffolds, to obtain structures with improved hydrophilicity and pharmacokinetics, as compared to peptidic, aromatic, or polymeric scaffolds. We were able to perform regioselective oxidation of mono-, di-, trisaccharides and cyclodextrins. We started preparing multimannosides, galactosides, and lactosides, synthesized by coupling the corresponding alkynyl glycosides to the azido modified oligosaccharides by CuAAC. We obtain a considerable gain in affinity to model lectins as Concanavalin A and PNA. Some of our glycoclusters were able to inhibit galectins 1 and 3. Afterward, we synthesized other glycoclusters exposing thiosugars, showing higher stability towards glycosyl hydrolases than O-glycosides. S-galactosides, S-lactosides, 3-deoxy-S-lactosides, and analogs of dithiogalactoside (a commonly used galectin inhibitor) afforded new multivalent probes to analyze sugar-lectin interactions. We finally explored a supramolecular approach for the construction of multivalent architectures, able to interact with proteins, through the design and synthesis of thiolactose-based amphiphiles. Interestingly, two compounds which only differ in the length of the spacer connecting the sugar fragments to the scaffold showed different properties. In this presentation, we will present the synthesis and affinities of different glycoclusters, and we discuss the influence several parameters as the triazole moiety, the length of the spacer, the ability for crosslinking of our glycoclusters, and the self-assembling properties of sugarbased amphiphiles.
Fiona Haxho has special interest and expertise in the glycosylation of human cancer cells, specifically the transient modifications in sialylation patterns of malignant cells. Her research has shown that the removal of terminal sialic acid residues from growth factor receptors on the surface of malignant cells, including insulin receptors and, epidermal growth factor receptors among others, is highly regulated by and dependent on perquisite G protein-coupled receptor signaling. Drug targeting of these protein complexes, particularly neuraminidase-1 and GPCR, has important implications for the development of novel molecular therapies in the treatment of human cancer.
G protein-coupled receptors (GPCR) can participate in a number of signaling pathways, and this property led to the concept of biased GPCR agonism. Agonists, antagonists and allosteric modulators can bind to GPCRs in different ways, creating unique conformations that differentially modulate signaling through one or more G proteins. A unique neuromedin B (NMBR) GPCR-signaling platform controlling mammalian neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP9) crosstalk has been reported in the activation of the insulin receptor (IR) through the modification of the IR glycosylation. Here, we propose that there exists a biased GPCR agonism as small diffusible molecules in the activation of Neu1-mediated insulin receptor signaling. GPCR agonists bombesin, bradykinin, angiotensin I and angiotensin II significantly and dose-dependently induce Neu1 sialidase activity and IR activation in human IR-expressing rat hepatoma cell lines (HTC-IR), in the absence of insulin. Furthermore, the GPCR agonist-induced Neu1 sialidase activity could be specifically blocked by the NMBR inhibitor, BIM-23127. Protein expression analyses showed that these GPCR agonists significantly induced phosphorylation of IR? and insulin receptor substrate-1 (IRS1). Among these, angiotensin II was the most potent GPCR agonist capable of promoting IR? phosphorylation in HTC-IR cells. Interestingly, treatment with BIM-23127 and Neu1 inhibitor oseltamivir phosphate were able to block GPCR agonist-induced IR activation in HTC cells in vitro. Additionally, we found that angiotensin II receptor (type I) exists in a multimeric receptor complex with Neu1, IR? and NMBR in naï¿½ve (unstimulated) and stimulated HTC- IR cells with insulin, bradykinin, angiotensin I and angiotensin II. This complex suggests a molecular link regulating the interaction and signaling mechanism between these molecules on the cell surface. These findings uncover a biased GPCR agonist-induced IR transactivation signaling axis, mediated by Neu1 sialidase and the modification of insulin receptor glycosylation.
Dr Nilofer Qureshi has more than 40-years’ experience in LPS related research (> 150 publications), and has played a pioneering role in studies related to cholesterol biosynthesis, structure and biosynthesis of mycolic acids in Mycobacterium tuberculosis; purification, structure and biology of several LPS/lipid A; and the mechanisms by which LPS alters innate immunity via proteasomes and causes disease. She has expertise in designing lipid A molecules for use as adjuvants such as MPLA and RsDPLA, which is a powerful LPS antagonist in both in vitro and in vivo systems. She has recently developed a new cell model that explains how LPS or other agonists activate inflammatory processes in various cell types, based on proteasomes. Proteasomes are present in all cells and play an integral role on the activity of the immune system. The proteasome is a central regulator of macrophage function and inflammation involved in several diseases. This model will be useful for designing novel drugs for sepsis, diabetes, asthma, cancer, AIDS, and neurological disease based on modulating the activity of proteasomes. This research was supported by NIH grants.
Statement of the Problem: During pathogen-mediated inflammation, host peripheral blood mononuclear cells/monocytes/ macrophages (PBMC/MO/M??) play a critical role in exacerbating or resolving disease by (a) priming of naï¿½ve and resident cells for selective host responsiveness; (b) extensive release of cytokines, reactive oxygen species, and nitric oxide; and, (c) development of hyporesponsiveness (tolerance/refractoriness) where selective host responses are repressed. Mechanisms of bacteria-triggered development of diseases in patients provide a prototype example of each of the responses elicited to various extents during disease progression/resolution. It is well-established that the human host innate immune system has developed the capacity to recognize and respond immunologically to multiple bacterial structures, such as lipopolysaccharides (LPS), peptidoglycan, and CpG DNA. However, all cells behave differentially with respect to agonists. The mechanisms underlying the modulation of these immune cells with respect to LPS are not well defined and conflicting results are reported. The purpose of this study is to understand the role of LPS in innate immune responses, such as induction of growth factors, priming, proliferation, differentiation, cytokines, nitric oxide, autophagy and death in PBMC/MO/M?? based on the proteasome???s proteases (a complex that degrades key regulatory proteins). A novel model based on the function of subunits of the proteasome was developed in both human and murine cells, based on genomics, proteomics, and signaling pathways. Several hormones, dietary nutrients, and vitamins were also tested in PBMC/MO/M?? from normal and diseased subjects with surprising results. Conclusion & Significance: The innate immune response induced by LPS in human monocytes (huMO) and mouse M??, as described above is largely dependent on the change in composition/function of cellular proteasomes in cells. This novel information will lead to the development of drugs for sepsis, diabetes, asthma, cancer, AIDS, and neurological diseases, based on solid findings on the modulation of proteasomes in immune cells.
Rebecca Laposa has a strong interest in mitochondrial toxicity and mitochondrial drug delivery, particularly in cancer therapeutic strategies. She has interest and experience in translational pharmacology research with a focus towards drug development. At the level of the molecular biology of nucleic acids, she is interested in DNA and RNA damage, and DNA and RNA responses to those molecular insults.
Oleg N Tikhodeyev is the author of the original approach for resolving multiple ambiguities and contradictions in current genetic concepts. He has shown that the key source of such ambiguities and contradictions is the erroneous belief that the same genetic term (for example, mutation) is able to comprise both specific phenomenology and the underlying mechanisms (Tikhodeyev, 2015). This belief became widely accepted after 1952 when the hereditary role of DNA had been demonstrated. In modern genetic concepts, the terms describing molecular mechanisms should be clearly distinguished from those describing phenomenology because there is no strict correlation between phenomenology and molecular mechanisms.
Statement of the Problem: It has long been accepted that any hereditary factor in any organism is represented by DNA sequences. This idea became fundamental in molecular genetics and was implicitly transformed into the DNA theory of inheritance. All basic genetic terms (genotype, gene, allele, mutation, recombination, etc.) were considered as specific DNA sequences or their alterations. However, multiple examples of stable epigenetic inheritance lacking any distinctions in DNA sequences were recently discovered, and the most exciting among them is protein inheritance. Amyloid hereditary prions in fungi were considered as ???protein-only??? hereditary factors, which features were determined entirely by protein conformation. As a result, the principal question arises whether the DNA theory of inheritance is wrong or not. Methodology & Theoretical Orientation: Considering different variants of the same hereditary prion as prion alleles, we examined the molecular nature of such variety. Findings: To perpetuate stably in cell generations a certain prion allele requires two entities: a specific state of the prion protein, and the corresponding DNA sequence to provide reproduction of the prion particles. We name these entities as the DNA determinant and the epigenetic determinant, respectively. Thus, a certain prion allele is a bimodular hereditary system depending on both the DNA determinant and the epigenetic determinant. Alteration of any of these two determinants may result in the establishment of a novel prion allele. Moreover, similar regularities are characteristic to all other cases of epigenetic inheritance, irrespective to the underlying mechanisms. Conclusion & Significance: The hereditary role of DNA is fundamental for any known mechanisms of inheritance, including epigenetic. However, it becomes an element of a more complicated concept: in addition to ???DNA-only??? hereditary factors, various bimodular hereditary factors also exist.
Dr Robert Brosh Jr received his PhD in Biology from the University of North Carolina at Chapel Hill, MS in Biochemistry from Texas A&M University, and BS in Chemistry from Bethany College. He conducted postdoctoral training at the Laboratory of Molecular Genetics (NIA, NIH) before assuming his position as a Principal Investigator where he leads the Section on DNA Helicases. In 2015, Dr Brosh received the NIH-NIA Post Baccalaureate Distinguished Mentor Award and in 2017 the NIH-NIA Director’s Merit Award. He is Associate Editor of Ageing Research Reviews. He serves on several editorial boards including the Journal of Biological Chemistry, Aging, and Genes and international grant review boards including NIH, Italian Telethon, and German Research Foundation. He is a member of the American Federation of Aging Research National Scientific Advisory Council, lectures at Johns Hopkins University, and is involved in Outreach Programs to provide children hands-on learning opportunities in science.
Statement of the Problem: The Section on DNA Helicases, NIA-NIH investigates the roles of human DNA helicases in genomic stability, prompted by the findings of hereditary helicase disorders characterized by accelerated aging and cancer. Methodology & Theoretical Orientation: Building on our discovery of small molecule inhibitors of the WRN helicase defective in Werner syndrome, we are conducting studies with compounds to target human helicases for inhibition (or activation) in cell-based model systems as a novel approach to assign specific helicase functions in vivo that may pave the way for development of therapeutic strategies. Of particular interest are helicase-interacting compounds that accentuate the effects of anti-cancer drugs based on a DNA damaging paradigm in genetically defined mutant backgrounds. Development of preclinical models is underway to evaluate the efficacy of helicase-targeted drugs for personalized medicine. In a parallel effort, we are conducting RNA interference screens with isogenic helicase-deficient and helicase-proficient cell lines to identify novel interactions between human helicases and target genes. Conclusion & Significance: These studies are built upon the premise that DNA helicases exhibit a network of synthetic lethal interactions that may be exploited to overcome resistance to existing anti-cancer therapies.
Dr Anthony Berdis is an Associate Professor in the Chemistry Department at Cleveland State University. He is also Co-Founder and Chief Scientific Officer for Red5 Pharmaceuticals, LLC which develops novel agents targeting drug-resistant cancers. Dr Berdis received his PhD in Biochemistry from the University of North Texas. He performed an NIH sponsored postdoctoral fellowship under the direction of Professor Stephen J Benkovic at the Pennsylvania State University. Dr Berdis has published over 80 research papers and book chapters. His research has been funded by the National Institutes of Health, the National Science Foundation, and the Department of Defense.
Temozolomide is a DNA damaging agent that is a frontline therapeutic agent against brain tumors including glioblastoma (GBM). Unfortunately, resistance to temozolomide occurs frequently, thus limiting the overall utility of the agent. Resistance can be caused by a variety of different cellular defects. However, an emerging mechanism of resistance reflects the ability to DNA polymerases to misreplicate DNA lesions that are left unrepaired. In this study, we provide evidence that the therapeutic efficacy of temozolomide can be significantly increased by co-administration of an artificial nucleoside, denoted as 5-nitroindolyl-2???-deoxyriboside (5-NIdR), that efficiently and selectively inhibits the replication of DNA lesions generated by temozolomide. Conversion of 5-NIdR to the corresponding nucleoside triphosphate 5-NITP in vivo creates a potent inhibitor of several human DNA polymerases that can replicate damaged DNA. This inhibition accounts for the ability of 5-NIdR to synergize with temozolomide to increase apoptosis of tumor cells. In a murine xenograft model of GBM, treatment with temozolomide only delayed tumor growth while co-administering 5-NIdR with temozolomide caused complete tumor regression. Preliminary toxicology studies demonstrate that high doses of 5-NIdR do not produce the side effects typically observed with conventional nucleoside analogs such as fludarabine and gemcitabine. Collectively, these preclinical results demonstrate pharmacological proof of concept for the coordinate inhibition of translesion DNA synthesis as a strategy to improve chemotherapeutic responses in aggressive brain tumors.