München, Techn. Universiẗat, Diss., 2004.

Human Papillomaviruses (HPVs) are circular double-stranded DNA (dsDNA) viruses that infect human cutaneous and mucosal tissue. Most HPV infections are benign or cause only minor pathologies. However, infection with one of the ~15 high risk types of HPV is associated with a variety of head/neck and anogenital cancers. All told, HPV infection is thought to cause ~5% of all human cancers and cause ~275,000 deaths per year. Despite causing immense morbidity and mortality, many aspects of how HPV virions successfully establish infection in host cells remain poorly characterized. Infection begins with HPV virions binding the cell surface, where they are modified by the host protease furin. The HPV virions are then endocytosed by association with an unknown entry receptor(s). After endocytosis the HPV minor capsid protein L2 acts as a chaperone to ensure that the viral genome (vDNA) traffics from endosomes to the trans-Golgi network (TGN) and eventually the nucleus, where HPV replication occurs. En route to the nucleus, the L2/vDNA complex must translocate across limiting intracellular membranes. The details of these critical processes remain poorly characterized. In this work we investigate the viral and host factors involved in the penetration of host membranes by the HPV L2/vDNA complex. First, we elucidated many of the viral and host factors necessary for furin cleavage of L2. We also demonstrate that furin cleavage mediates the homo-oligomerization and membrane insertion of L2. Finally we demonstrate that complete translocation across the limiting membrane is dependent on host cell entry into mitosis. Overall this work provides novel insight into the molecular mechanisms used by HPV virions to breach host membranes and establish infection.

Class III Semaphorins (SEMA3) comprise a family of chemokines that have been implicated as negative regulators of axonal guidance, angiogenesis and tumor progression. It has been demonstrated previously that one SEMA3, SEMA3F, may have therapeutic potential in the treatment of cancer. When transfected with SEMA3F, the highly metastatic human melanoma cell line A375SM was found to exhibit a highly-encapsulated, avascular phenotype with limited metastasis. Members of SEMA3 are regulated on many levels, including proteolytic processing. SEMA3F, like other SEMA3, is expressed as a 100 kD proprotein that is seen to be processed in vitro and in vivo to 95 and 65 kD isoforms. This has been largely attributed to furin-like endoproteases on the basis of furin inhibition studies. However, currently available small chemical or peptide inhibitors against the family of subtilisin/kexin-type proprotein convertases (PCSK), to which furin belongs, do not have good selectivity between PCSKs. Cleavage of SEMA3 to 65 kD have been shown to have differing effects. SEMA3A loses its ability to repel sympathetic ganglia and SEMA3E reverses its phenotype from chemorepulsant to chemoattractant for developing vasculature following cleavage. In order to further develop therapeutic strategies based on SEMA3F, it is therefore critical to better understand the proteolytic regulation of this molecule. In this study, it is shown that digest of purified SEMA3F with purified recombinant human furin does not result in proteolytic cleavage and suggested that the cleavage of SEMA3F to a 65 kD isoform may be mediated by other members of the PCSK family.

Introduction: Mammalian cell recombinant bone morphogenetic protein (rBMP) synthesis is reported to be poor. The BMP pro-domain may be involved in folding, stability and secretion. Objectives: Investigate the effect of inhibition of pro-domain cleavage on rhBMP-2 production. Methods: CHO cells transfected with human BMP-2 (hBMP-2) were cultured in the presence of the proprotein convertase inhibitor 9DR in short (multi-well) and long-term (bioreactor) cultures. Mature and proBMP secretion was measured by ELISA and characterized by Western blot. BMP activity was determined by C2C12 bioassay. Results: 9DR significantly enhanced the yields of both pro- and mature hBMP-2 in short and long-term cultures, without any negative effects on cell growth or viability. The rhBMP-2 was biologically active. ProBMP-2 could be converted by exogenous furin treatment into mature BMP-2 as shown by Western blot. Conclusions: 9DR increases rhBMP-2 production and is a simple, but effective way to enhance the yield of active, mature BMP-2 in CHO cells.

Introduction: Mammalian cell recombinant bone morphogenetic protein (rBMP) synthesis is reported to be poor. The BMP pro-domain may be involved in folding, stability and secretion. Objectives: Investigate the effect of inhibition of pro-domain cleavage on rhBMP-2 production. Methods: CHO cells transfected with human BMP-2 (hBMP-2) were cultured in the presence of the proprotein convertase inhibitor 9DR in short (multi-well) and long-term (bioreactor) cultures. Mature and proBMP secretion was measured by ELISA and characterized by Western blot. BMP activity was determined by C2C12 bioassay. Results: 9DR significantly enhanced the yields of both pro- and mature hBMP-2 in short and long-term cultures, without any negative effects on cell growth or viability. The rhBMP-2 was biologically active. ProBMP-2 could be converted by exogenous furin treatment into mature BMP-2 as shown by Western blot. Conclusions: 9DR increases rhBMP-2 production and is a simple, but effective way to enhance the yield of active, mature BMP-2 in CHO cells.

The skyrocketing costs of prescription medicine in developed countries and their lack of availability in developing countries are the most challenging problems of human health. Primary reasons for such high cost are fermentation-based production, expensive purification methods, the need for low temperature storage and transportation and the delivery through sterile injections. Most of these expenses could be minimized or eliminated when therapeutic proteins are expressed and orally delivered via plant cells. Chloroplasts have the machinery to fold complex and biologically active eukaryotic proteins in the soluble chloroplast stromal compartment. Protein expression through chloroplast transformation system offers a number of advantages over nuclear transformation such as a high level of transgene expression (up to 47% of the total soluble protein), due to the presence of 10,000 copies of the transgene per cell, which is uniquely advantageous for oral delivery of adequate amounts of the therapeutic protein or vaccine antigen. It is also an environmentally friendly approach due to effective gene containment and lack of transgene expression in pollen since the chloroplast genome is maternally inherited. To study receptor-mediated oral delivery of therapeutic proteins using the transmucosal carrier cholera toxin B subunit (CTB), a CTB-GFP fusion protein separated by a furin cleavage site was expressed via the tobacco chloroplast genome and used as a visible marker. Site specific integration of the transgene was confirmed by PCR analysis. Southern blot analysis confirmed homoplasmy. Immunoblot analysis confirmed the expression of both the monomeric as well as the pentameric forms of CTB-GFP in transgenic plants. Expression levels of upto 21.3% were obtained and the functionality of the CTB-GFP pentamers was confirmed by an in vitro GM1 binding assay. GFP was seen in the intestinal mucosa, liver and spleen of mice orally fed with CTB-GFP expressing leaves, while CTB was detected only in the intestinal cells. Intestinal macrophages and dendritic cells stained positive for both the CTB as well as GFP. These results suggest successful cleavage of the foreign protein from the transmucosal carrier and its delivery to various organs. These investigations should facilitate the development of a novel cost-effective oral delivery system for plant-derived therapeutic proteins.

No description available.

Release Date: October 2017 Anemia of chronic inflammation (ACI) is a condition that develops in a setting of chronic immune activation. ACI is characterized and triggered by inflammatory cytokines and the disruption of iron homeostasis. Hepcidin, a small peptide hormone, is the master regulator of iron homeostasis, and rapidly responds to iron supply and demand. Under conditions of chronic inflammation, hepcidin is elevated, and alters the way that iron is absorbed and disrupted throughout the body, resulting in disrupted iron homeostasis through inhibition of the iron exporter protein ferroportin. Anemia arises when insufficient erythropoietic activity combined with inadequate iron supply abrogates the healthy development of mature red blood cells (RBCs). The proprotein convertase (PC) known as furin is a serine protease capable of cleaving peptide precursors into their active state. Furin is critical for normal activation of proteins and enzymes but recently, furin has been implicated in critical roles within cancers, viral and pathogenic infections, and arthritis through activating precursors novel to the disease type. Furin has previously been identified as being the sole PC responsible for generating active hepcidin. Hepcidin is initially synthesized as a larger precursor protein, before undergoing furin cleavage. Furin is known to form mature, bioactive hepcidin, with the removal of this pro-region. Our discovery of a novel regulatory site on Furin has led to potent inhibition using small molecules. This inhibition is shown with the use of in vitro fluorogenic assays, in vivo cell tissue cultures, and within an animal model of ACI. Our results demonstrate that in using these small molecules we can decrease hepcidin levels even in the presence of potent inflammatory cytokines. The inhibition of hepcidin by these small molecules causes an increase in stably expressed ferroportin on cell surfaces and the restoration of the ability to mobilize iron from storage tissues and absorption from the diet. The ability to "fine-tune" inhibition of furin in targeting its allosteric site along with its catalytic domain designates these small-molecule inhibitors as promising therapeutics for treatment of diseases ranging from Alzheimer's and cancer to anthrax and Ebola fever.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18905号 / 医科博第61号 / 新制||医科||4(附属図書館) / 31856 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 河本 宏, 教授 武田 俊一, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Membrane-type 5 matrix metalloproteinase (MT5-MMP) is unique among MMP family members as it is predominately expressed in the CNS. Its expression is ubiquitous during brain development and restricted to regions of neurogenesis and neuroplasticity in the adult. MT5-MMP is a mediator of pericellular proteolysis and is thought to have a functional impact on neurite outgrowth. The studies presented in this work were designed to examine MT5-MMP expression in cultured NT2 cells, a model of newogenesis and neuronal differentiation, and in adult neurogenic brain regions. We further sought to overexpress MT5-MMP and test the hypothesis that it plays a role in substrate-specific cell motility.MTS-MMP mRNA was expressed in NT2 cells and was significantly higher in differentiated neuronal hNT cells. MT5-MMP cDNA cloned from NT2 cells unexpectedly revealed a novel sequence (MTS-MMPvar) which was 92% homologous with the published MT5-MMP and was characterized by a 162 bp deletion. Both transcripts could be identified in NT2, hNT and adult human hippocampus. The newly cloned MT5-MMPvar cDNA translated into an approximately 52 kDa protein as seen in in vitro expression studies. Using an MTS-MMPvar specific antibody designed to span the 162 bp deletion, MT5-MMPvar protein could be detected in NT2 cells and these protein levels increased in their neuronal counterparts, hNT cells. MT5-MMPvar protein was also expressed in adult human hippocampal tissue. MT5-MMPvar protein was shown to be expressed in a murine region of neurogenesis and plasticity, suggesting the existence of a murine homolog of this variant.Based on bioinformatic analysis, the MT5-MMPvar transcript was predicted to lack a sufficient signal peptide and to remain a Type-I membrane protein. This computer assisted modeling suggests that the most significant functional implication of MTS-MMPvar sequence variations is to affect its direction into the ER for processing. Functional studies using COS-7 cells genetically modified to overexpress MT5- MMPvar demonstrated no difference in cellular motility compared to parental or vector control cells. Preliminary studies show MT5-MMPvar expression in COS-7 cells associated with perinuclear structures and the cell membrane. Adult human neural progenitor cells stimulated to differentiate into immature neurons demonstrated MT5-MMPvar expression associated with the cell membrane and process outgrowths. This work has identified a novel transcript variant of the human MTS-MMP gene and shown that the protein product of this gene is significantly higher in differentiated NT2 cells. This, combined with preliminary results suggesting MT5- MMPvar cellular redistribution in more mature cell types, indicates a role for MTSWvar in neural differentiation and function.