| 1. |
- Digre, Andreas, 1987-
(författare)
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Implications of Heparan Sulfate and Heparanase in Inflammatory Diseases
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Heparan sulfate (HS), an unbranched sulfated carbohydrate chain, and the HS-degrading enzyme heparanase play important roles in physiological and pathological processes during all stages of life, from early embryogenesis to ageing. Accumulated information shows that HS and heparanase are involved in inflammatory processes and associated diseases, e.g. rheumatoid arthritis (RA) and Alzheimer’s disease.In this thesis I have investigated the role of HS and heparanase (Hpa) in inflammatory-related pathologies. In the first project, Hpa overexpressing mice (Hpa-tg) were induced with a murine model of RA. We found a pro-inflammatory role of Hpa through enhancing the activity of T-cells and innate immune cells, which contributed to an augmented severity of clinical symptoms in the Hpa-tg mice.In my second project, we revealed co-current interaction of heparin with both ApoA1 and SAA of HDL isolated from plasma of inflamed mouse. Mass spectrometry analysis indicated close proximity of ApoA1 and SAA on the HDL surface, providing a molecular and structural mechanism for the simultaneous binding of heparin to apoA1 and SAA.In my third project, we investigated the role of Hpa in AA amyloid formation and resolution in mice in a model of AA-amyloidosis. We found a similar degree of amyloid formation in Hpa-KO mice compared to the wildtype control mice, but the resolution process was faster in Hpa-KO mice. The rapid clearance of deposited SAA in Hpa-KO mice was associated with upregulated expression of matrix metalloproteases. The results suggest an associated function of ECM proteases with heparanase in the process of AA amyloid resolution.In my fourth project, we found that overexpression of heparanase impaired inflammation associated beta amyloid (Aβ) clearance in the brain of an Alzheimer’s disease mouse model. Examination of the cytokine profile of brain lysates revealed an overall lower inflammatory reaction in the double transgenic (tgHpa*Swe) mice compared to single APP-tg (tg-Swe) mice in response to LPS-induced inflammation.
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| 2. |
- Batool, Tahira
(författare)
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Heparan sulfate dependent cell signaling and associated pathophysiology : Implications in tumorigenesis and embryogenesis
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Heparan sulfate proteoglycans (HSPGs) consist of a protein core to which several linear, negatively charged heparan sulfate (HS) chains are covalently attached. HSPGs are expressed on the cell surface and in the extra-cellular matrix (ECM) where they have diverse biological functions, for example co-receptor functions. The diverse functions of HS are linked to structural variability of the polysaccharide. In this thesis, I investigated HS structure-function relationship by using different cell and animal models of one HS-biosynthetic enzyme, glucuronyl C5-epimerase (Hsepi) and one enzyme responsible for post synthetic modification, heparanase.Deletion of Hsepi in mice resulted in neonatal lethality, with multiple organ defects, indicating the importance of HS in embryogenesis. Up-regulated expression of heparanase is found in most human tumor tissues, correlating with increased metastatic potential and decreased survival of cancer patients.In the first project, I focused on the effects of HS on cancer associated cell signaling and found that heparanase overexpression attenuated TGF-β1 stimulated Smad phosphorylation in tumor cells because of increased sulfation degree and turnover rate of HS.Heparanase role in cancer progression has led to clinical trials where inhibition of heparanase activity is currently being evaluated as a potential cancer treatment. Heparin, a HS-related polysaccharide, is being used to inhibit heparanase activity. In my second project, we studied the effect of low molecular weight heparin (LMWH) on cisplatin resistance of ovarian cancer cells (A2780cis). LMWH treatment of A2780cis cells reduced Wnt-activity in these cells and consequently reduce the drug resistance.In paper III, we continued exploring the HS/heparanase role in cancer by using heparanase overexpressing mice (Hpa-tg). We found Lewis Lung Carcinoma (LLC2) cells showed faster growth, bigger tumors and more metastasis in the Hpa-tg mice as compared to wild-type (WT) mice, because of suppressed antitumor immunity in the Hpa-tg mice.In paper IV and V, we studied the structure-function relationship of HS by using Hsepi-/- mice model. Hsepi-/- results in HS-chains lacking IdoA, which makes the chain rigid and consequently affects its co-receptor function. Skeletal malformation in Hsepi-/- mice, led us in paper IV to investigate bone morphogenic protein (BMP), an important signal molecule during embryogenesis and known to interact with HS. We found upregulation of a number of BMPs and expression of P-smad1/5/8, but reduced expression of inhibitory Smads and Gremlin1 in the Hsepi-/- MEF cells. The study indicated that the developmental defects in Hsepi mice could be contributed by a higher BMP signaling. In paper V we investigated the lung of the Hsepi-/- mice. The distal lung of 17.5 days old embryos remained populated by epithelial tubules, because of impaired differentiation of type I cells of the lungs. Potential mechanisms behind the failure of type I cell formation was identified to be reduced vascularization and a sustained signaling of Smad pathways.
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| 3. |
- Beckman Sundh, Ulla, 1953-
(författare)
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Studies on Phosphohistidine Phosphatase 1 : What? Where? Why?
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Phosphohistidine phosphatase 1 (PHPT1) is a small protein, consisting of 125 amino acids, that catalyzes the dephosphorylation of histidine but does not have any activity towards other phosphorylated amino acids. PHPT1 was identified in 2002, and is so far the only mammalian histidine phosphatase known, but still little is known about its physiological role. No mammalian histidine kinases have hitherto been identified.Phosphorylation is one of the most important ways in which the structure and activity of a protein may be changed after translation. Proteins are phosphorylated on the side chain of amino acid residues. When a hydroxyl is phosphorylated the result is a phosphoester and when a nitrogen is phosphorylated the result is a phosphoamidate. Histidine may be phosphorylated on either of the two nitrogens of the imidazole ring of the side chain. The resulting phosphoamidate bond is labile and rich in energy, which makes histidine phosphorylation highly reversible and flexible. However, histidine phosphorylation is less studied than that of the phosphoesters due to the acid lability of the phosphoamidate bond.The work described in this thesis was focused on further elucidating the physiological role of PHPT1. Amino acid residues of importance for the activity of PHPT1 were identified, and mutants with decreased phosphatase activity were produced. These mutants have been used in studies on the function of PHPT1. By using immunohistochemical methodology the localization of PHPT1 in both mouse and human tissues was determined, with mainly similar results. A general finding was that expression of PHPT1 was high in epithelial cells with short turnover time, indicating that PHPT1 may have an important role in proliferating cells. We have also developed a comparatively fast and simple screening method for determination of PHPT1 activity. Since research in this field has been hampered by the lack of efficient and practical methodology, hopefully this new method will be an asset in search of inhibitors for PHPT1, which in turn may be used for detection of the elusive mammalian histidine kinases, the finding of which may give major breakthroughs in the field.
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| 4. |
- Jia, Juan, 1979-
(författare)
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Structure and functions of heparan sulfate/heparin – Importance of glucuronyl C5-epimerase and heparanase
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Heparan sulfate (HS) and heparin are linear polysaccharide chains covalently O-linked to serine residues within the core proteins, so called HS proteoglycans (PGs) or heparin PG. HSPGs are produced by almost all mammalian cells and known to play important roles in developmental processes, physiological and pathological conditions; whereas heparin PG is produced by mast cells and best known as an anticoagulant in clinic. Biosynthesis of HS/heparin occurs in Golgi compartment and involves many enzymes, one of which is glucuronyl C5-epimerase (Hsepi) that catalyzes the conversion of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA). Heparanase is an enzyme involved in metabolism of HS; it cleaves the linkage between GlcA and glucosamine residues in HS/heparin chains. Heparanase is expressed essentially by all cells and found up-regulated in many metastatic tumors. This thesis focuses on the structure and functions of HS/heparin through studies on the implications of Hsepi and heparanase. My study demonstrated that the modification catalyzed by Hsepi is critical for HS-dependent function of growth factors, especially FGF2; heparanase is involved in regulation of HS biosynthesis and matrix metalloproteinases expression; moreover, my experimental data demonstrated the functions of heparin in mast cells, showing cleavage of heparin by heparanase contributes to modulation of protease storage in mast cells.
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| 5. |
- Noborn, Fredrik, 1978-
(författare)
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Heparan Sulfate Dependent Mechanisms of Amyloidosis
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A common theme in amyloid disorders is the deposition of disease-specific protein aggregates in tissues. Amyloid proteins bind to heparan sulfate (HS), a sulfated glycosaminoglycan, and HS has been found to promote the aggregation process. The present work relates to HS mediated mechanisms of amyloidosis, particularly transthyretin (TTR) amyloidosis, AA-amyloidosis and Alzheimer’s disease (AD). TTR is a transport protein present in the blood and cerebrospinal fluid, which under unclear circumstances can deposit as amyloid in the myocardium of elderly individuals. Examination of cardiac tissue from a 70 year old patient with reported cardiomyopathy reveald co-deposition of TTR amyloid and HS. Studies revealed that HS promotes TTR fibrillization through interaction with a basic motif in the protein. Empolyment of a cell model demonstrated that cell surface HS mediates internalization of TTR, an effect likely facilitated by HS-binding to the basic motif on TTR. Collectively, HS-TTR interactions at the cell surface may have dual outcomes, resulting in either fibrillization or internalization, respectively. During inflammatory conditions, serum amyloid A (SAA), an acute-phase protein associated with the high-density lipoprotein (HDL), can assemble into insoluble amyloid fibrils, causing AA-amyloidosis. We found that HS structures exceeding 12-14 sugar units in length separates SAA from HDL and induces subsequent aggregation of the polypeptide. Our result proposes a novel role for HS in AA-amyloidosis in which a critical length of HS is required for separation of SAA from HDL. Late-onset AD patients show reduced ability to clear cerebral amyloid-β (Aβ) aggregates, a pathological hallmark of the disease. Althought the pathway of Aβ clearance is still unclear, several cell-surface receptors are implicated in Aβ internalization. We found that ApoE facilitated Aβ uptake through interactions with HS-proteoglycans and low-density lipoprotein receptor-related protein 1. The ApoE interaction with Aβ likely promotes Aβ clearance in the brain, but, if unbalanced, may contribute to the pathology of AD. These findings are in accord with the concept of HS as a promoter of amyloid protein aggregation, but also point to more complex relationship.
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| 6. |
- Hjertström, Elina, 1981-
(författare)
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Roles of Heparan Sulfate in Amyloid-β Pathology and Hypoxia
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Heparan sulfate (HS) is a highly sulfated polysaccharide expressed on the cell surface and in the extracellular matrix, interacting with a large number of proteins. HS is implicated in human diseases, including different types of cancer and amyloid diseases such as Alzheimer's disease (AD). The aims of this thesis were to gain deeper insights into AD and cancer progression by elucidating the roles of HS in amyloid-β (Aβ) pathology and hypoxia. The toxic Aβ-peptide is a key molecule in AD due to its ability to aggregate and form amyloid plaques in the brains of diseased patients. It has been reported that HS accumulates with Aβ in these amyloid plaques. We have found that HS is differentially accumulated with Aβ species within the amyloid plaques in the brains of AD patients. We also identified that the HS in the plaques originated from glial cells. Further, we investigated the role of HS in Aβ toxicity using cell models that either lack HS or express abnormal HS. The results show that cell surface HS mediates Aβ internalization and cytotoxicity. Upregulation of heparanase, an endo-glucuronidase that specifically cleaves HS chains, in human cancers increases the potential of tumor cells to metastasize. Spalax, an animal model for hypoxic tolerance, expresses high levels of heparanase. Analysis of HS from different Spalax organs revealed a high sulfation degree and an atypical domain structure, likely modulated by high heparanase expression in the organs. Cells cultured under hypoxic conditions showed a similar HS domain structure and had an increase in heparanase mRNA. We propose that hypoxia-induced heparanase expression is relevant for tumor progression, a process often associated with oxygen deficiency. Altogether, the findings in this thesis are important for future development of therapeutics aiming at interfering with HS functions in AD and cancer.
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