Sub-communities within this community

Collections in this community

Recent Submissions

  • Characterization of Hic-5 in Cancer Associated Fibroblasts: A Role in Extracellular Matrix Deposition and Remodeling

    Turner, Christopher; Goreczny, Gregory (2017)
    Hic-5 (TGFβ1i1) is a focal adhesion scaffold protein that has previously been implicated in many cancer-related processes. However, the contribution of Hic-5 during tumor progression has never been evaluated, in vivo. In Chapter 2 of this thesis, I crossed our Hic-5 knockout mouse with the MMTV-PyMT breast tumor mouse model to assess the role of Hic-5 in breast tumorigenesis. Tumors from the Hic-5 -/-;PyMT mouse exhibited an increased latency and reduced tumor growth. Immunohistochemical analysis of the Hic-5 -/-;PyMT tumors revealed that the tumor cells were less proliferative. However isolated tumor cells exhibit no difference in growth rate. Surprisingly, Hic-5 expression was restricted to the tumor stroma. Further analysis showed that Hic-5 regulates Cancer Associated Fibroblast (CAF) contractility and differentiation which resulted in a reduced ability to deposit and reorganize the extracellular matrix (ECM) in two-and three-dimensions. Furthermore, Hic-5 dependent ECM remodeling supported the ability of tumor cells to metastasize and colonize the lungs.The molecular mechanisms by which CAFs mediate ECM remodeling remains incompletely understood. In Chapter 3 of this thesis, I show that Hic-5 is required to generate fibrillar adhesions, which are specialized structures that are critical for the assembly of fibronectin fibers. Hic-5 was found to promote fibrillar adhesion formation through a newly characterized interaction with tensin1, a scaffold protein that binds to β1 integrin and actin. Furthermore, this interaction was mediated by Src-dependent phosphorylation of Hic-5 in two and three-dimensional matrix environments to prevent β1 integrin internalization and subsequent degradation in the lysosome. This work highlights the importance of the focal adhesion protein, Hic-5 during breast tumorigenesis and provides insight into the molecular machinery driving CAF-mediated ECM remodeling.
  • Bundling of cytoskeletal actin by the formin FMNL1 contributes to celladhesion and migration

    Blystone, Scott; Miller, Eric (2018)
    Metastasis is one of the leading causes of death in the world, affecting thousands every year. This is especially true of breast cancer, which can often result in the formation of secondary metastatic sites in the lung, liver, and bone marrow. There are many aspects to metastasis and an innumerable amount of molecular, biochemical, and cellular interactions contribute to its pathology. The ability of primary tumor cells to disseminate from the primary tumor, degrade the basement membrane, invade through the ECM, and eventually intravasate across the endothelial cell lining of the circulatory system or lymphatics requires a plethora of proteins, all working together in concert to achieve this. Nowhere in the cell is this more apparent than the actin cytoskeleton.Locomotion of cells requires several alterations in the actin cytoskeleton component of the cellular machinery. Generally speaking, cells must be able to polarize, form protrusions, adhere to the substratum, translocate, and then retract their tail, repeating this process as they continue to navigate to their destination. While there are many underlying aspects to this activity, spatiotemporal rearrangements of the actin cytoskeleton are key to the successful cellular motility. The mechanics behind dynamic actin cytoskeletal modifications are varied and complex, demonstrating the requirement for a variety of actin-associated, regulatory proteins.A crucial family of proteins involved in this process is the formin family of proteins. Formins are a relatively “new” group of actin modifiers which possess the unique ability to modify and generate linear actin filaments. While the members of this protein family all share some of the same actin modifying processes, many of these proteins also have functions exclusive to themselves. As a result, research into this field has blossomed and several novel features of different formins have been identified. Furthermore, alternative splice isoforms of several formins are often expressed in a variety of cell types, with specific functions attributed to each.The formin FMNL1 was originally identified in cells of a myeloid lineage and for many years was mostly thought to be involved in leukocyte adhesion and migration. Indeed, our lab has characterized many of the functions of this protein in both human and murine macrophages. However, as a result of the work in this dissertation, we have generated sufficient evidence suggesting that FMNL1 not only plays a role in breast cancer migration, but also exhibits functions unique to a specific alternative splice isoform of this protein.Our work on FMNL1 has pushed the field of study into this protein family in new directions. Herein, we have demonstrated that all three alternative splice isoforms of FMNL1 are expressed in a variety of cell types and the FMNL1ɣalternative splice isoform distinguishes itself from these isoforms via its ability to bundle linear actin filaments. Additionally, our data indicates that this is accomplished independently of the trademark FH2 domain, often thought to be the essential component of all formins. More specifically, we have identified a unique amino acid sequence in the C-terminal region of this isoform that most likely regulates this function. As a result, we have not only identified a potential therapeutic target for the treatment of metastasis via inhibition of cellular locomotion, but also pushed the field of formin research into a novel direction by providing insight which may foster new hypotheses and challenge classical theories regarding the relationship between formins and actin.
  • BRAIN SPECIFIC NEURAL EXTRACELLULAR MATRIX EXPRESSION AND MODIFICATIONS IN NEUROLOGICAL DISEASE AND DISORDERS

    Matthews, Rick; Dwyer, Chrissa (2013)
    The central nervous system (CNS) is extraordinarily complex in both structure and function. The neural extracellular matrix (ECM) is one of the key classes ofmolecules that regulates thedevelopment of the CNS and maintains its structure and function in the adult.Thereby understanding the function of the neural ECMis key to understanding the CNS. The neural ECM is composed of several nervous-system specificproteins, which are hypothesized to uniquely contribute to the defining physiological functions of the CNS. However,work in this area has been hindered by the highly complex molecular properties of the neural ECM, which stem from alterations in expressionand modifications (resulting from glycosylation and proteolytic cleavage) of its constituents. Further defining mechanisms that alter the expression and modifications of neural ECM constituents are critical to fully understanding its complex array of functions. Often in neuropathologies, the neural ECM undergoes dynamic changes providing a valuable tool to further understand its function andthe opportunity to explore its contribution to disease pathology and utility as a therapeutic target. The work presented herein investigates the role of altered expression of the nervous-system specific ECM constituent, Brain Enriched Hyaluronan Binding (BEHAB)/ brevican(B/b), in glioma,and altered glycosylation of the nervous system enriched ECM constituent, RPTPζ/phosphacan, in O-mannosylrelated congenital muscular dystrophies (CMDs). Our work suggests that increased expression of B/b in the glioma tumor microenvironment (TEM) contributes to the pathological progression of these tumors, and reducing its expression is a valuable therapeutic strategy. Additionally, our work evaluates the transcriptional regulatory mechanisms leading to increases inB/b expression in glioma and highlights the potential value of these mechanisms as therapeutic targets. Our work also identifies the absence of O-mannosyl linked carbohydrates on RPTPζ/phosphacan in the brains of CMD models and suggests that altered glycosylation of RPTPζ/phosphacan may have a role in the neuropathologies underlying these disorders. Overall this work provides valuable insight intothe molecular complexities of the neural ECM stemming from changes in the expression and glycosylation of its constituents and furthers our understanding of its function in the normal CNS and in neuropathologies.
  • PHOSPHORYLATION AND UBIQUITINATION REGULATE PROTEIN PHOSPHATASE 5 ACTIVITY AND ITS PROSURVIVAL ROLE IN KIDNEY CANCER

    Mollapour, Mehdi; Dushukyan, Natela (2018)
    Protein Phosphatase 5 (PP5) is a serine/threonine phosphatase known to regulate many essential cellular functions including steroid hormone signaling, stress response, proliferation, apoptosis, and DNA repair. PP5 is a knownco-chaperone of the molecular chaperone heat shock protein 90 (Hsp90), and its regulation of Hsp90aidswiththe proper activation of Hsp90 clients and withsteroid hormone signaling.Hsp90 is also one of the strongest activators of PP5, as it releases the auto-inhibition of PP5 by interacting with the N-terminal tetratricopeptide repeat (TPR) domain of PP5. Our lab has recently shown that PP5 is phosphorylated at T362, and that this phosphorylation acts as an “on switch” resultingin the hyperactivation of PP5. Misregulation of this key phosphatase has been shown to aid in the tumor progression of ER-dependent and independent breast cancer. Elevated PP5 levels have also been linked to colorectalcancer, hepatocellular carcinoma (HCC), lymphoma, and prostate cancer. The work presented here reveals the pro-survival role that PP5 plays in kidney cancer. Clear cell renal cell carcinomas (ccRCC) are most often driven by mutations in the von Hippel-Lindau tumor suppressor (VHL). The data in this thesis shows that VHL binds and multi mono-ubiquitinates PP5 at two lysine residues K185 and K199. This post-translational modification negatively regulates PP5 likean “off switch” and ultimately leads to its degradation bythe proteasome. Mutations in the VHLgene that result in inactive mutants or a lack of VHL protein expression lead to ccRCC tumors. The data in this thesis shows that these VHL-nulltumors become dependent on elevated levels of PP5, and that both PP5 knockdown and inhibition lead to cancer cell death. The data further shows that the decrease in PP5 activity in VHL-null cells results in the induction of the extrinsic apoptotic pathway with a dramatic increase in the cleavage of PARP and caspases 3, 7, and 8.
  • POST-TRANSLATIONAL REGULATION OFCO-CHAPERONES AFFECTS HSP90 DRUG SENSITIVTY IN CANCER

    Mollapour, Mehdi; Dunn, Diana (2017)
    Heat Shock Protein-90 (Hsp90) is a molecular chaperone critical to thestability and activity of over 200 proteins known as “clients” including many oncogenes. Hsp90 chaperone function is linked to its ability to hydrolyze ATP and Hsp90 drugs inhibit its activity leading to the degradation of clients, thus making Hsp90 an attractive target for cancer therapy. The Hsp90 chaperone cycle is fine-tuned by another group of proteins called co-chaperones. They modifythe cycle, allowing Hsp90 to chaperone different pools of clients. Post-translational modifications (PTM) of Hsp90 and its co-chaperones can also regulate the chaperone cycle, and affect Hsp90 drug sensitivity. Here it is shownthat c-Abl kinase phosphorylates Y223in the co-chaperoneAha1, promotingits interaction with and stimulation of Hsp90 ATPase activity. Pharmacologic inhibition of c-Abl prevents the Aha1-Hsp90 interaction thereby, hypersensitizing cancer cells to Hsp90 inhibitors.Another co-chaperone of Hsp90, protein phosphatase-5 (PP5), mediates thede-phosphorylation of the co-chaperone Cdc37which is an essentialprocessfor the activation of kinase clients. The crystal structure of phospho-Cdc37 bound to the catalytic domain of PP5revealed elements of substrate specificity within the phosphatase cleft. Hyperactivityandhypoactivity of PP5 increasedHsp90 binding to its inhibitor, providing insight into increasingthe efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.PP5 is autoinhibited by intramolecular interactions that can be activated by anumber of cellular factors, includingHsp90. Casein kinase-1δ (CK1δ)-mediated phosphorylation of T362-PP5, was identified as an integral step for PP5 activation, independent of binding to Hsp90. Additionally, the tumor suppressor von Hippel-Lindau (VHL), the substrate recognition component of the VCB-E3-ubiquitin ligase, was found to interact with and multi-monoubiquitinate K185/K199-PP5 for proteasomal degradationin an oxygen-independent manner. Furthermore, VHL-deficient clear cell renal cell carcinoma (ccRCC) cell lines or patient tumors exhibit elevated PP5 levels. Down-regulation of PP5 caused apoptosis inccRCC cells, suggesting a prosurvival role for PP5 in ccRCC.Thisevidence suggests that inhibition of the enzymes that target and catalyze the PTM of Hsp90 and co-chaperones can act synergistically with Hsp90 inhibitors, providingnovel therapeutic strategiesto enhance the efficacy of Hsp90 inhibitors in cancer cells.
  • MITOCHONDRIAL ELECTRON TRANSPORT CHAIN ACTIVITY IN SYSTEMIC LUPUS ERYTHEMATOSUS

    Perl, Andras; Doherty, Edward (2014)
    Systemic lupus erythematosus (SLE) is an autoimmune disorder, characterized by T cell and B cell dysfunction. SLE mitochondria have been shown to be dysfunctional with increased mass, mitochondrial potential, decreased ATP, elevated reactive oxygen species (ROS) and reactive nitrogen species (RNS) concentrations, and altered Ca2+ stores. Drug treatments that target the mitochondria have shown efficacy in treating SLE. Here we have investigated electron transport chain (ETC) activity in SLE, to better understand the causes of mitochondrial dysfunction in SLE. We have found that mitochondrial complexes I and IV of the ETC have elevated respiration in SLE compared to healthy controls after both overnight resting and anti-CD3/CD28 stimulation. We have also shown that SLE complex I is resistant to NO inhibition of respiration. SLE peripheral blood lymphocytes (PBL) have increased S-nitrosylation (SNO) while immunoprecipitated complex I had decreased SNO of proteins compared to healthy controls. The drug Nacetylcysteine (NAC) was able to inhibit complex I activity in SLE, and was found to reduce the amount of complex I protein NDUFS3 after 15 minutes as measured by western blotting. These results have led us to the conclusion that SLE mitochondrial complex I is in an active form which is resistant to SNO and is driving the production of ROS and RNS that are associated with SLE. The drug NAC is able to inhibit complex I respiration which may have therapeutic efficacy by reducing the ROS and RNS stress in SLE.
  • Preclinical Development of Anti-Cancer Drugs from Natural Products.

    Huang, Ying; Sun, Qing (2014)
    Cancer has been and will continue to be the common concern in the United States and worldwide. As a conventional treatment to fight cancer, new anti-cancer drugs with more efficiency and less toxicity are extremely required. In this study, we have identified two novel compounds with anti-cancer properties from two traditional Chinese medicinal plants. One is Lappaol F that was extracted from the seeds of the plant Actium Lapp L., which has been used in China for centuries as anti-viral and anti-bacterial medicine. Another is M-9 that was extracted from the stem of Marsdenia tenacissima,a plant that has been applied to treat inflammation and cancer in China. Our results showed that Lappaol F inhibited cancer cell growth by regulating a series of cell cycle related proteins and inducing cell cycle arrest at G1 and/or G2 phase. p21 played a critical role in Lappaol F-induced cyclin B1 and cyclin-dependent kinase 1 (CDK1) suppression as well as G2arrest. Lappaol F also induced cell death in a number of cancer cells through caspases activation. Lappaol F-mediated cell growth inhibition was p53-independent. Notably, results from animal studies showed that Lappaol F effectively inhibited tumor growth in vivo, while being well tolerated by the mice. Thus, Lappaol F has a strong potential to be developed as a novel anti-cancer chemotherapeutic. Our studies showed that M-9 successfully sensitized several tumor cells but not non-tumorigenic cells to paclitaxel (Taxol) treatment. Additionally, M-9 reversed chemotherapeutic resistance in a number of multidrug resistant cells. Further results suggested that M-9 functioned, at least to a certain extent, via inhibiting drug efflux by competitively binding to P-glycoprotein (P-gp), a protein that accounts for multidrug resistance. Importantly, results from the in vivostudies demonstrated that M-9 strongly enhanced Taxol-induced growth suppression against xenografts derived from HeLa cells. Moreover, mice tolerated the treatment of Taxol and M-9 well. Therefore, M-9 is a novel chemosensitizer candidate to overcome P-gp-mediated multidrug resistance. Taken together, our studies provide a solid basis for further development of these two compounds as anti-cancer remedies.
  • RELN AS A CANDIDATE GENE FOR AUTISM SPECTRUM DISORDER (ASD)

    Howell, Brian; Lammert, Dawn (2017)
    Autism spectrum disorder (ASD) affects approximately 1 in 45 people, and is characterized by deficits in social communication and repetitive behaviors. Sequencing advancements have enabled the identification of numerous candidate genes, but precisely how these genes contribute to ASD remains largely unknown. RELNis consistently implicated as a candidate gene for autism. The encoded secreted glycoprotein, Reelin is important for proper brain developmental and postnatal synapse function. Here we examine the molecular and cellular consequences of the de novo RELNmutation R2290C. This mutation falls in a conserved arginine-amino acid-arginine (RXR) motif that is found within the Reelin subrepeat structure. Several other ASD patient mutations fall with in this consensus and all examined reduce Reelin secretion. Based on this we tested two hypothesis: (1) that the mutations reduce Reelin signaling and (2) that they have a gain-of-function consequence, such as ER stress. Using an engineered cell line with a heterozygous RELNR2290C mutation and the RELN Orleans (Orl) mouse line that produces nearly full length Reelin that is defective for secretion, we found evidence for both increased Dab1 and increased PDIA1 expression. Since, like most genes implicated in ASD RELNlikely acts in a multifactorial manner, we investigated whether second site mutations might contribute to ASD-related behaviors. Towards this end we crossed the heterozygous Orl and Shank3b mice to model two hits that are present in at least one ASD proband. We found that the resulting double heterozygousmice had impaired socialization and altered ultrasonic vocalizations. Furthermore, forebrain and cerebellar lysates showed increased PSD-95, identifying a potentially common mechanism and therapeutic target for ASD. These studies are the first to investigate the biological relevance of RELNcoding mutations in ASD.
  • A matter of life and death: human cytomegalovirus induction of monocyte survival and differentiation into macrophages through manipulation of the PI3K/Akt pathway

    Chan, Gary; Cojohari, Olesea (2017)
    Human cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus infecting up to 80% of the US population and reaching 100% seroprevalence in many parts of the world. In mostindividuals HCMV infection is usually asymptomatic. In contrast, in immunodeficient or immunonaive people, such as transplant recipients and the developing fetus, the virus is a major cause of morbidity and mortality. During a primary infection, HCMVcan spread very effectively in the body infecting many organ types and monocytes are believed to be the principal cell type responsible for HCMV dissemination throughout the body. Monocytes, however, are naturally programmed to undergo apoptosis after 48h in the circulation and are not permissive for viral replication. Our lab has shown that in order to combat these biological hurdles, HCMV promotes survival of these short-lived cells past their 48h “viability gate”. Besides inducing survival, the virus also mediates the differentiation of monocytes into macrophages skewed towards an M1 pro-inflammatory phenotype with select M2 anti-inflammatory features, which are long-lived cells, permissive for viral replication. However, the mechanisms used by HCMV to concomitantly induce survival and macrophage differentiation -two linked but separate processes, are not fully understood. The studies in this thesis reveal that upon binding and entry, HCMV initiates a survival program in monocytes by inducing a rapid and sustained activation of the PI3K/Akt pathway, which isdifferent from that induced by myeloid growth factors. Moreover, after inducing cellular survival across the 48-h viability gate, the virus also employsthe PI3K/Akt pathway to regulate caspase 3 activation which mediatesthe atypical M1/M2 polarization. Our work suggests that virus not only makes use of the PI3K/Akt pathway, but manipulates it at multiple levels toallow for viral-specific downstream functional changes.Deciphering how the virus uniquely maneuvers signaling pathways in monocytes to drive their survival and differentiation might allow us to develop new treatments targeting HCMV-infected monocytes and preventing viral spread and disease.
  • The Role of TET Methylcytosine Dioxygenase 2 in Myeloid Malignancies

    Mohi, Golam; Nath, Dipmoy (2017)
    TET methylcytosine dioxygenase 2 (TET2) catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an intermediate stage in the DNA demethylation processthat controls the transcription of genes. TET2 is highly expressed in the hematopoietic system and is suggestedto regulate the maintenance and differentiation of hematopoietic stem/progenitors. Mutations in TET2 has been found in different hematological malignancies including Acute Myeloid Leukemia (AML), Chronic Myelomonocytic Leukemia (CMML), Myeloproliferative Neoplasms (MPN), Myelodysplastic Syndrome (MDS), etc. However, the mechanisms by which TET2 controls hematopoiesis and contributes to myeloidmalignancies remainunknown. The direct targets of TET2 have not been elucidated yet. In order to identify the direct targets of TET2, we have performed chromatin immunoprecipitation using TET2 specific antibody followed by genome-wide sequencing. We have found enrichment in binding of TET2 in the promoters of SHP1, SOCS3 and PLZF among other targets. Interestingly, the expression of these genes is also significantly downregulated in the hematopoietic progenitors of TET2 deficient mice. Furthermore, we have found that CMML patients with TET2 mutations also have decreased expression of these genes. Knockdown of TET2 resulted in downregulation of these genes in leukemic Molm14 and murine Ba/F3 cells. Conversely, overexpression of TET2 in monocytic U937 cells increased the expression of these genes. Using methylation specific PCR, we also have observed increased methylation in the promoters of SHP1, SOCS3 and PLZF in TET2 deficient Molm14 and Ba/F3 cells. Also, using methyl/hydroxymethyl-DNA immunoprecipitation, we observed an increase in the 5mC level and decrease in the 5hmC level in the promoter region of these genes suggesting that TET2 directly regulates the expression of these genes by regulating the methyl and hydroxymethyl level of the promoter of these genes. Although loss of function of TET2 has been associated with multiple hematopoietic malignancies, TET2 is most commonly mutated in CMML with almost 50% patients bearing TET2 mutations. Interestingly, TET2 mutations are frequently associated with CBL mutations in CMML. In order to assess the concurrent effects of TET2 and CBL deficiencies, we generated TET2 CBL double knockout mice. We observed that simultaneous deletion of TET2 and CBL resulted in increased leukocytes and neutrophil and enhanced splenomegaly compared to control mice. The double knockout mice showed increase in the granulocyte macrophage progenitors and a significant expansion of the stem progenitor cell population. The overall survival of these mice also reduced substantially. It suggests that concurrent deletion of TET2 and CBL increased the severity of the CMML like disease in mice and thus TET2 and CBL deletion may cooperate in the pathogenesis of CMML. Although most studies suggest a tumor suppressor function of TET2, we also have found a tumor promoter function, especially in MLL rearranged leukemia. We have found that knockdown of TET2 resulted in decreased proliferation in MLL-AF9 positive Molm14 leukemia cells and murine Ba/F3 MLL-AF9 expressing cells. Conversely overexpression of TET2 significantly increased the proliferation level of Molm14 cells. In orderto understand the in vivo role of TET2 in MLL-AF9 mediated leukemia, we performed a retroviral BMT experiment. Whereas the expression of MLL-AF9 in wild type BM resulted in marked increase in WBC and NE and splenomegaly, the deletion of TET2 reduced the white blood cell and neutrophil count and also caused reduction in the spleen size. MLL-AF9 overexpression resulted in the increase in hematopoietic stem/progenitor cells and granulocyte macrophage progenitors and granulocyte/monocyte precursors which was significantly reduced in TET2 KO MLL-AF9 mice. There was a significant reduction of the hematopoietic colony formation ability mediated by MLL-AF9 in TET2 KO mice. The overall survival of the knockout mice was markedly improved compared to the control MLL-AF9 mice. Together these results suggest a tumor promoter role of TET2. Taken together, all the results indicate a dual role of TET2 in myeloid leukemia.
  • ANTIVIRAL ACTIVITY AND MECHANISM OF ACTION OF A NOVEL URACIL ANALOG FOR VARICELLA-ZOSTER VIRUS

    Moffat, Jennifer; DE, CHANDRAV (2015)
    The alphaherpesvirus varicella-zoster virus (VZV) is widespread in humans. VZV causes primary and recurrent diseases that are preventable with live vaccines and are treatable with antiviral drugs. New antiviral drugs for varicella-zoster virus (VZV) with increased potency are needed, especially to prevent post-herpetic neuralgia. The purpose of this project was to evaluate β-L-1-[5-(E-2-Bromovinyl)-2-(hydroxymethyl)-1,3-dioxolan-4-yl)] uracil (L-BHDU) and 5′-O-valyl-L-BHDU for efficacy, safety, resistance, and mechanism of action in three models of VZV replication: primary human foreskin fibroblasts (HFFs), skin organ culture (SOC) and in SCID-Hu mice with skin xenografts. We found that L-BHDU and valyl-L-BHDU were safe and effective against VZV in culture and in a mouse model. Herpes simplex virus Type 1 was also sensitive to LBHDU in cultured cells. The mechanism of action of L-BHDU and its effect on drugdrug interactions were not known. Given its similar structure to brivudine (BVdU), we addressed whether L-BHDU, like BVdU, inhibits 5-fluorouracil (5-FU) metabolism. LBHDU did not interfere with 5FU metabolism, indicating that L-BHDU is a safer drug than BVdU. However, L-BHDU antagonized the activity of acyclovir (ACV), BVdU and foscarnet (PFA) in cultured cells, which was due to competition for phosphorylation by VZV thymidine kinase (TK). The mechanism of action of L-BHDU was studied by evaluating its activity against related α-herpesviruses and by analyzing resistant VZV strains. VZV strains resistant to L-BHDU (L-BHDUR) were cross-resistant to ACV and BVdU but not to PFA and cidofovir (CDV). Whole genome sequencing of L-BHDUR strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q) domains of VZV TK. The purified L-BHDUR TKs were enzymatically inactive and failed to phosphorylate the drug. In wild type VZV- infected cells, L-BHDU was converted to L-BHDU mono- and diphosphate forms; cells infected with L-BHDUR virus did not phosphorylate the drug. We also investigated whether addition of nucleosides reversed LBHDU inhibition of VZV in dividing and quiescent HFFs. Excess thymidine and uridine, but not purines, in proportion to L-BHDU restored VZV replication only in dividing cells, suggesting that the active form of L-BHDU interfered with pyrimidine biosynthesis. Like other herpesviruses, VZV infection induced thymidine triphosphate (dTTP) in confluent cells while L-BHDU treatment decreased the dTTP pool. Some herpesviruses raise dNTP pools by inducing cellular enzymes. However, VZV infection did not increase cellular thymidylate synthase (TS) expression to facilitate viral replication. Furthermore, the active form of L-BHDU did not interfere with cellular metabolism, suggesting a viral target. Further studies are required to identify the target(s) of L-BHDU active form(s).
  • The p53-Zn2+ Energy Landscape and Metallochaperone Hypothesis

    Loh, Stewart; Blanden, Adam (2017)
    p53 is a tumor suppressor protein found mutated in essentially half of human cancers, and dysfunctional in nearly all human cancers. Each DNA-binding domain of the protein contains a critical tetrahedrally coordinated Zn2+. In this work, we present a quantitative thermodynamic model describing the energetics of the p53-Zn2+ interaction, as well as the mechanism of action of a new class of therapeutic compounds we call synthetic zinc metallochaperones (ZMC) that restore proper structure and function to many mutant p53s by delivering Zn2+ to the protein in the cell. We combine recombinant protein expression and in vitrobiophysical characterization with cell biology, molecular biology, medicinal chemistry, and live cell imaging to address these issues. Our model for both the mechanism of action of ZMCs and the p53-Zn2+interaction are broadly based on the Metallochaperone Hypothesis originally proposed by our group in 2010. We find that the core tenants of the Metallochaperone Hypothesis are accurate, and have expanded that model to quantitatively describe the link between p53-Zn2+ binding and protein stability noted for decades in the field. We find that at physiological temperature and Zn2+ concentrations, wild-type p53 has a folding energy of ~0 kcal mol-1, and as such is exquisitely sensitive to inactivation by mutation, and rapidly changes the fraction folded in response to changes in Zn2+ concentration. We demonstrate that ZMCs are ionophores, transport Zn2+ from the extracellular space into cells, and rescue mutant p53 by increasing the intracellular free Zn2+ concentration. This increase in Zn2+ stabilizes the mutant proteins via the same mechanism previously described for substrate stabilization of enzymes, and is only seen in a "Goldilocks Zone" of Zn2+ concentrations and ZMC Kds. This presents a fundamentally new way to interact with and reactivate mutant p53s, and raises questions about the potential for biological exploitation of this interaction for signaling or other functions.
  • Role of RPTPzeta/phosphacan in the neural extracellular matrix

    Matthews, Rick; Eill, Geoffrey (2020)
    The mammalian brain is the most structurally and functionally complex system in biology. In order to carry out diverse functions such as thought and cognition, neurons in the brain must properly differentiate, make millions of functional interconnections, and incredibly be able to maintain those interconnections while retaining plasticity and the ability to learn throughout its lifetime. In order for neurons to carry out these complex functions, they must have an intricate relationshipwith their extracellular environment, which provides numerous molecules, such as growth factors and neurotransmitters, and physical cues to initiate critical downstream signaling cascades. In the central nervous system (CNS), the neural extracellular matrix (ECM) largely organizes this extracellular environment, and as such, is implicated in a multitude of neuronal functions. Not only can it serve as a physical barrier, the neural ECM is shown to regulate neuronal cell differentiation, migration, synaptogenesis, and maintain the mature state by restricting plasticity and neurite outgrowth. Of particular import, a subset of the neural ECM, the perineuronal net (PNN), is implicated to regulate neuronal plasticity in the CNS. PNNs were historically believed to be critical for restricting experience dependent plasticity in the brain but more recently shown to regulate several forms of learning and memory, in addition to multiple neurological diseases. However, despite these implicated functions, PNNs comprise only a fraction of the total ECM in the CNS. A majority of the neural ECM is derived from the diffuse ECM, a structure that is ubiquitously expressed throughout the CNS. As the diffuse ECM is very similar in molecular composition and structure to PNNs, it is difficult to specifically target PNNs for functional and mechanistic studies.As such, a better toolset is needed to differentiate the contributions ivbetween PNNs and the diffuse ECM in regulating plasticity and learning and memory. To develop thistoolset, there is a great need to better understand PNN molecular composition and structure. Therefore, the purpose of this work was to provide critical insight into the molecular composition and structure of PNNs soas to better understand its function in the CNS. In Chapter 2, using multiple ECM genetic knockout models, we show compelling evidence of an additional PNN anchor that importantlylays the groundworkforfuture functional studies. Specifically, we suggestchondroitin sulfate proteoglycan (CSPG) phosphacan, secreted isoform of receptor protein tyrosine phosphatase zeta (RPTPζ), iscritical for PNN structure as it partially anchors PNNs to the neuronal surface through cooperation with tenascin-R. Additionally, as the neural ECM is involved in numerous neurological diseases, in Chapter 3, we investigated the function of major ECM component, RPTPζ, in a group of O-mannosyl related congenital muscular dystrophy with associated brain abnormalities (CMD). Our data suggest a possible role of RPTPζ in proper cortical lamination in a CMD mouse model. Interestingly, we found evidence of a novel O-mannosyl substrate in the developing brain that could critically contribute to the underlying deficits of CMD. In conclusion, the neural ECM, once previously disregarded in the field, is becoming a novel source to understand mature mammalian brain function and disease, but more work is needed to better differentiate the specific roles of its substructures.
  • THE ROLE OF DENGUE VIRUS NON-STRUCTURAL PROTEIN 1 IN DISEASE PATHOGENESIS

    King, Christine; Endy, Timothy; Barbachano-Guerrero, Arturo (2020)
    Dengue virus (DENV) causes an estimated 390 million infections worldwide annually, with severe forms of disease marked by vascular leakage and an over reactive inflammatory response. Endothelial cells (EC) are directly responsible for vascular homeostasis and are highly responsive to circulating mediators but are not commonly infected. Mast cells (MC) are potent cells of the innate immune system that play an important role in EC biology and inflammatory responses. DENV encodes 10 proteins; with only one, the non-structural protein 1 (NS1), secreted from infected cells and accumulating in the blood of patients.NS1 has been implicated in the pathogenesis of vascular permeability, but the mechanism is not completely understood. Using a complementary array of in vitroassays and disease relevant ECs and MCs, we described the possible roles for NS1 in dengue disease pathogenesis. Using microscopy and immunoblotting we observed that ECs internalize NS1 into endosomes, where it accumulates and is degraded overtime. Transcriptome and pathway analysis defined changes in global gene expression in ECs that are associated with cell dysfunction. We observed that NS1 induced an increase in multicellular rearrangements and a decrease in barrier function in ECs. We demonstrated that NS1-dependent activation of the p38 MAPK pathway controls the changes in EC permeability in vitro. Further, we discovered iiithat ECs and MCs respond to NS1 by secreting a specific array of proinflammatory cytokines and chemokines that may contribute to the cytokine storm in dengue disease. Finally, we found that NS1 internalization can mediate the uptake of bound antibodies into ECs. Together, these results suggest a vasoactive and proinflammatory role for DENV NS1 that may participate in the development of severe symptoms in dengue disease. The observed functions of NS1 could lead to the discovery of new therapeutic targets in dengue disease.
  • Expression and Function of Paxillin Genes in Zebrafish: A Role in Skeletal Muscle Development

    Turner, Chris; Amack, Jeffrey; Jacob, Andrew (2017)
    Paxillin is a key component of the Integrin adhesion complex, which regulates cellular signaling events in response to extracellular matrix interactions. Although the roles for Paxillin in cell migration have been extensively studied, less is understood about its role in vertebrate development. Depletion of Paxillin from mouse embryos results in early lethality due to impaired cardiovascular development and function, necessitating the development of alternative vertebrate genetic models for examining the role of Paxillin during embryogenesis. Zebrafish have emerged as an experimental vertebrate model amenable to genetic manipulation. The work compiled herein first characterizes the expression profiles for Paxillin genes in zebrafish, and then describes the embryonic phenotypes observed upon mutation of these genes. The identification of two Paxillin genes in zebrafish, pxnaand pxnb, provided new insight into the evolution of this gene family in the Teleost lineage. Both overlapping and unique expression profiles for these genes during zebrafish embryogenesis were uncovered. While both genes are expressed in developing skeletal muscle, pxnawas restricted to the notochord during earlier stages of embryogenesis and pxnbwas expressed in the developing heart. Targeted mutation of either gene alone did not impair embryonic development, suggesting partial functional redundancy between each gene during embryogenesis. Accordingly, combined mutations in pxnaand pxnbrevealed defects during the development ofseveral embryonic tissues. In particular, skeletal muscle morphogenesis iiiwas perturbed in these double mutant embryos. Further characterization revealed that Paxillin genes in zebrafish serve to regulate embryonic myotome shape and proper extracellular matrix composition during muscle development. The amount of Laminin was reduced, while the abundance of Fibronectin persisted, during myotome morphogenesis in Paxillin double mutant embryos. In addition, a role for cytoskeletal contractility in regulatingsubcellular localization of Paxillin in developing skeletal muscle was established. Defects in the development of the cardiovascular system were also apparent in Paxillin double mutant embryos, and future work will focus on characterizing these in further detail. Altogether, this work provides a new vertebrate model to use for understanding the role of Paxillin during embryonic development, and uncovers an unrecognized role for Paxillin in establishing the extracellular matrix of skeletal muscle.
  • EVALUATION OF THE ROLE OF THE CENTRAL PAIR IN CHLAMYDOMONAS REINHARDTII FLAGELLAR MOTILITY

    Mitchell, David; Brandon, Smith (2013)
    Cilia and flagella are essential for the function of nearly all eukaryotes. This organelle is made up of nine outer doublet microtubules and two central singlet microtubules to form the canonical (9+2) ciliary structure. Cilia and flagella use this structure, as well as several protein complexes, such as the outer and inner dynein arms, the radial spokes, and the proteins that decorate the central pair to propagate the bending that produces motion. Flagellar motion is highly regulated, and each of these structures is necessary to regulate the dynein arms that generate the motile force. The central pair is one of the least understood of these structures. To date there are two major impediments hindering our understanding of the central pair: a lack of understanding as to how distinct central pair structures work in concert, and a general lack of available central pair mutant strains in the model organism Chlamydomonas reinhardtii. In order to further our understanding of how the central pair functions I have used multiple strategies. Firstly I have used previously characterized central pair mutants to study both structural interactions within the central pair and how the double mutant affects motility regulation. Secondly I provide evidence that a potential central pair mutant, H2, is indeed a central pair mutant and affects the C2b projection. Lastly I will attempt to characterize a new Chlamydomonas mutant, 10B5. Together these analyses will demonstrate that double mutants can have an additive effect on the structure of the central pair, and that double central pair mutants do not appear to suppress one another, but are at least ivepistatic to the most severe phenotype. I will also show evidence that 10B5 is not a central pair mutant, but with further study it may offer new insight into motilityregulation.
  • ANTIVIRAL ACTIVITY AND MECHANISM OF ACTION OF A NOVEL URACIL ANALOG FOR VARICELLA-ZOSTER VIRUS

    Moffat, Jennifer; DE, CHANDRAV (2015)
    The alphaherpesvirus varicella-zoster virus (VZV) is widespread in humans. VZV causes primary and recurrent diseases that are preventable with live vaccines and are treatable with antiviral drugs. New antiviral drugs for varicella-zoster virus (VZV) with increased potency are needed, especially to prevent post-herpetic neuralgia. The purpose of this project was to evaluate β-L-1-[5-(E-2-Bromovinyl)-2-(hydroxymethyl)-1,3-dioxolan-4- yl)] uracil (L-BHDU) and 5′-O-valyl-L-BHDU for efficacy, safety, resistance, and mechanism of action in three models of VZV replication: primary human foreskin fibroblasts (HFFs), skin organ culture (SOC) and in SCID-Hu mice with skin xenografts. We found that L-BHDU and valyl-L-BHDU were safe and effective against VZV in culture and in a mouse model. Herpes simplex virus Type 1 was also sensitive to LBHDU in cultured cells. The mechanism of action of L-BHDU and its effect on drugdrug interactions were not known. Given its similar structure to brivudine (BVdU), we addressed whether L-BHDU, like BVdU, inhibits 5-fluorouracil (5-FU) metabolism. LBHDU did not interfere with 5FU metabolism, indicating that L-BHDU is a safer drug than BVdU. However, L-BHDU antagonized the activity of acyclovir (ACV), BVdU and foscarnet (PFA) in cultured cells, which was due to competition for phosphorylation by VZV thymidine kinase (TK). The mechanism of action of L-BHDU was studied by evaluating its activity against related α-herpesviruses and by analyzing resistant VZV viii strains. VZV strains resistant to L-BHDU (L-BHDUR ) were cross-resistant to ACV and BVdU but not to PFA and cidofovir (CDV). Whole genome sequencing of L-BHDUR strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q) domains of VZV TK. The purified L-BHDUR TKs were enzymatically inactive and failed to phosphorylate the drug. In wild type VZV- infected cells, L-BHDU was converted to L-BHDU mono- and diphosphate forms; cells infected with L-BHDUR virus did not phosphorylate the drug. We also investigated whether addition of nucleosides reversed LBHDU inhibition of VZV in dividing and quiescent HFFs. Excess thymidine and uridine, but not purines, in proportion to L-BHDU restored VZV replication only in dividing cells, suggesting that the active form of L-BHDU interfered with pyrimidine biosynthesis. Like other herpesviruses, VZV infection induced thymidine triphosphate (dTTP) in confluent cells while L-BHDU treatment decreased the dTTP pool. Some herpesviruses raise dNTP pools by inducing cellular enzymes. However, VZV infection did not increase cellular thymidylate synthase (TS) expression to facilitate viral replication. Furthermore, the active form of L-BHDU did not interfere with cellular metabolism, suggesting a viral target. Further studies are required to identify the target(s) of L-BHDU active form(s).
  • Biological importance of TIMP-2 phosphorylation on MMP-2 activity

    Bourboulia, Dimitra; Bullard, Renee (2016)
    Matrix metalloproteinases (MMPs) are proteolytic enzymes that are secreted from the cell and play an important role in embryonic development and tissue remodeling. In cancer, MMPs are hyperactive, promoting degradation of the ex-tracellular matrix. Enhancement of MMP proteolytic activity allows tumor cells to migrate and invade surrounding tissues, increasing the chance of metastasis. Tissue inhibitor of metalloproteinases (TIMPs) are also known to act extracellu-larly, and are the endogenous inhibitors of MMPs. To inhibit the protease activi-ty of MMPs, the N-terminus of the TIMP protein binds to the catalytic domain of MMP at a ratio of 1:1. Studies from our lab have found that TIMP-2 is phosphor-ylated on three tyrosine residues, and this phosphorylation increases the inter-action with MMP-2. This is the first time that phosphorylation of TIMP-2 has been reported. Fascinatingly, the proto-oncogene tyrosine kinase c-Src was found to phosphorylate TIMP-2. This is significant in that c-Src has not yet been shown to act extracellularly, and there are no details within the current lit-erature describing how this protein may function outside of the cell. In this the-sis, we usedmammalian cells as a model to decipher whether TIMP-2 phosphor-ylation wasable to occur extracellularly,as well as the effect that phosphoryla-tion of TIMP-2 hadon its functionto both inhibit/activate MMP-2. We found that(1) c-Src is able to phosphorylate TIMP-2 extracellularly in conditioned me-vidia; and (2) phosphorylation of TIMP-2 enhances its function of inhibiting MMP-2 proteolytic activity, as well as assisting in the activation of pro-MMP-2. Our results suggest the presence of anovel mechanismin whichphosphoryla-tion of TIMP-2is able to regulate the extracellular environment through en-hanced interaction with MMP-2. The information gained from this research couldlead to development of novel therapies that use phosphorylated TIMP-2 as a means of decreasing cellular migration and invasion with the overall goal of preventing metastasis.
  • Mechanisms of aseptic loosening in total knee arthroplasty

    Cyndari, Karen (2017)
    Introduction: Cemented Total Knee Arthroplasty (TKA) is the gold standard of care for end-stage, multi-etiologic arthritis. While the longevity of these devices may now reach or even surpass 15 years in service, a minority (~10%) will fail prematurely due to a process called aseptic loosening. Historically, this process has been attributed to an inflammatory reaction against wear debris from the TKA polyethylene (PE) insert. However, we have previously estimated supraphysiologic fluid shear stress (FSS) (exceeding 900 Pa) at the cement-bone interface of cemented joint replacements, and examined this as a possible alternative cause of increased osteoclast activity. Methods: We analyzed the cement-bone interlock of tibial and femoral components from en-bloc, postmortem-retrieved, non-revised TKAs to explore the process of loss of fixation, prior to any clinical loosening. For the tibial components, we used a novel protocol wherein whole undecalcified bone+PMMA cement segments from the proximal tibia were embedded in Spurr’s resin and thinly sectioned. Polarized light microscopy was used to identify and quantify co-located PE debris. Using a novel bioreactor developed by our lab called the Multi-Well Fluid Loading (MFL) System, we examined static, subphysiologic, physiologic, and supraphysiologic FSS on RAW 264.7 osteoclast activity and morphology, with and without PE particle treatment. Results: We found no association been the amount or presence of PE debris and the amount or location of loss of interlock in retrieved TKAs. FSS up to 17 Pa increased the ability of osteoclasts to resorb mineral, and FSS up to 4.4 Pa induced the formation of larger osteoclasts. FSS and fluid shear rate interacted together to increase the area of actin rings, while PE treatment increased the number of actin rings and TRAP production. FSS up to 4.4 Pa decreased expression of Ctsk and Il1a, but PE co-treatment abolished this effect. Conclusions: These results indicate there may be alternative factors leading to aseptic loosening apart from PE debris. We demonstrated that osteoclasts are mechanosensitive and able to adjust activity, morphology, and gene expression based on FSS. Further, PE interferes with osteoclast gene downregulation in response to FSS, indicating PE could be a potentiator of osteoclast activity or differentiation.

View more