| 1. |
- Buijs, Jos, et al.
(författare)
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Localized changes in the structural stability of myoglobin upon adsorption onto silica particles, as studied with hydrogen/deuterium exchange mass spectrometry
- 2003
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Ingår i: Journal of Colloid and Interface Science. - 0021-9797 .- 1095-7103. ; 263:2, s. 441-448
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Tidskriftsartikel (refereegranskat)abstract
- A new method is presented for monitoring the conformational stability of various parts of a protein that is physically adsorbed onto nanometer-sized silica particles. The method employs hydrogen/deuterium (H/D) exchange of amide hydrogens, a process that is extremely sensitive to structural features of proteins. The resulting mass increase is analyzed with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Higher structural specificity is obtained by enzymatically cleaving the adsorbed proteins prior to mass spectrometric analysis. The mass increases of four peptic fragments of myoglobin are followed as a function of the H/D exchange time. The four peptic fragments cover 90% of the myoglobin structure. Two of the peptic fragments, located in the middle of the myoglobin sequence and close to the heme group, do not show any adsorption-induced changes in their structural stability, whereas the more stable C- and N-terminal fragments are destabilized. Interestingly, for the N-terminal fragment, comprising residues 1–29, two distinct and equally large conformational populations are observed. One of these populations has a stability similar to that in solution (−23 kJ/mol), whereas the other population is highly destabilized upon adsorption (−11 kJ/mol).
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| 2. |
- Danfelter, Mikael
(författare)
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Cartilage destruction - Release of type IX collagen in joint disease
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cartilage is of vital importance for the function of joints. It is characterized by a prominent extracellular matrix (ECM) that is produced and maintained by relatively few cells of one type, the chondrocyte. The cartilage ECM consists of collagen fibrillar networks, proteoglycan aggregates, and a number of other molecules united in numerous interactions. This work focused at identifying catabolic events leading to cartilage destruction and functional impairment and developing tools to detect such events in patients. In the first part, using a model for cartilage degradation in disease, N-terminal fragments of type IX collagen was found to be released at an intermediate time point. The two most abundant fragments were further studied and their novel C-terminals identified. These two fragments comprise most of the NC4 domain and the combined NC4 + COL3 domains, respectively. Furthermore, the identified cleavage sites were shown to be induced by matrix metalloproteinase 13 (MMP-13). In the second part, the potential degradation of type IX collagen in human cartilage was investigated. It was shown that two similar type IX collagen fragments were released from cartilage explants upon MMP-13 digestion. The novel C-terminal of the fragment comprising the NC4 domain was identified. The cleavage site was found to differ between human and bovine type IX collagen. An antiserum raised against the human C-terminal neoepitope confirmed the cleavage and was used to develop an immunoassay (inhibition ELISA). This assay was used to detect elevated levels of the neoepitope in sera from arthritis patients compared to controls, indicating that this cleavage occurs in joint pathology.
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| 3. |
- Danfelter, Mikael, et al.
(författare)
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Fragmentation of proteins in cartilage treated with IL-1. Specific cleavage of type IX collagen by MMP-13 releases the NC4 domain.
- 2007
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 282:51, s. 36933-36941
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Tidskriftsartikel (refereegranskat)abstract
- Degradation of bovine nasal cartilage induced by interleukin-1 (IL-1) was used to study catabolic events in the tissue over 16 days. Culture medium was fractionated by two-dimensional electrophoresis (isoelectric focusing and SDS-PAGE). Identification of components by peptide mass fingerprinting revealed released fragments representing the NC4 domain of the type IX collagen {alpha}1 chain at days 12 and 16. A novel peptide antibody against a near N-terminal epitope of the NC4 domain confirmed the finding and indicated the presence of one of the fragments already at day 9. Mass spectrometric analysis of the two most abundant fragments revealed that the smallest one contained almost the entire NC4 domain cleaved between arginine 258 and isoleucine 259 in the sequence -ETCNELPAR258-COOH NH2-ITP-. A larger fragment contained the NC4 domain and the major part of the COL3 domain with a cleavage site between glycine 400 and threonine 401 in COL3 (-RGPPGPPGPPGPSG400-COOH NH2-TIG-). The presence of multiple collagen {alpha}1 (IX) N-terminal sequences demonstrates that the released molecules were cleaved at sites very close to the original N terminus either prior to or due to IL-1 treatment. Matrix metalloproteinase 13 (MMP-13) is active and cleaves fibromodulin in the time interval studied. Cartilage explants treated with MMP-13 were shown to release collagen {alpha}1 (IX) fragments with the same sizes and with the same cleavage sites as those obtained upon IL-1 treatment. These data describe cleavage by an MMP-13 activity toward non-collagenous and triple helix domains. These potentially important degradation events precede the major loss of type II collagen.
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| 4. |
- Feng, Haoyu, et al.
(författare)
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Extracellular matrix in disc degeneration.
- 2006
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Ingår i: Journal of Bone and Joint Surgery. American Volume. - 1535-1386. ; 88:Suppl 2, s. 25-29
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Tidskriftsartikel (refereegranskat)abstract
- The extracellular matrix of the intervertebral disc structures contains many molecules also found in cartilage. The extremely polyanionic proteoglycans play a central role, particularly in the nucleus, by creating an osmotic environment leading to retention of water and ensuing resistance to deformation—important for the resilience of the tissue. Another major structural entity particularly important in the anulus is the network of collagen fibers; fibril-forming collagen 1 is a major constituent. The collagen fibrils in the anulus are largely oriented in sheets around the nucleus. A number of molecules present in the matrix regulate and direct the collagen fibril assembly by interacting with the collagen molecule and also the formed fibril. Several of these molecules bind by one domain to the collagen fiber and present another functional domain to interact either with other fibers or with other matrix constituents. In this manner the collagen fibers are cross-linked into a network that provides tensile strength and distributes load over large parts of the anulus. Diminished function in these cross-bridging molecules will lead to loss of mechanical properties of the collagen network and result in an impaired ability of the anulus to resist forces delivered by compression of the disc and particularly the nucleus. A different network abundant in the disc and in other load-bearing tissues is based on the beaded filaments of collagen 6. The basic building block is a tetramer of two pairs of antiparallel collagen-6 molecules arranged such that two N-terminal ends of collagen 6 are exposed at either end of the unit. Further assembly occurs both by end-to-end and side-to-side associations. This process is catalyzed by both biglycan and decorin, where the combined effect of direct binding of the core protein to the collagen-6 N-terminal globular domain and the presence of the glycosaminoglycan side chain is essential. These ligands are bound at the same site in complexes extracted from the tissue and then also have one bound molecule of matrilin-1, 2, or 3, in turn bound to a collagen fiber, a procollagen molecule, or an aggrecan. Interactions at the cell surface provide signals to the cells with regard to the conditions of the matrix. Such interactions include binding by matrix components to various receptors at the cell surface. Remodeling of the matrix takes place in response to various factors. An early event in disease is degradation of aggrecan by the members of the ADAMTS (a disintegrin-like and metalloprotease with thrombospondin motifs) family and degradation of molecules important in maintaining the collagen network.
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| 5. |
- Hansson, Ola, et al.
(författare)
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Transcriptome and proteome analysis of soleus muscle of hormone-sensitive lipase-null mice
- 2005
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Ingår i: Journal of Lipid Research. - 1539-7262. ; 46:12, s. 2614-2623
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Tidskriftsartikel (refereegranskat)abstract
- Hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization in adipocytes, has been demonstrated also in skeletal muscle. To gain further insight into the role and importance of HSL in skeletal muscle, a transcriptome analysis of soleus muscle of HSL-null mice was performed. A total of 161 transcripts were found to be differentially expressed. Increased mRNA levels of fructose-1,6-bisphosphatase, fructose-2,6-bisphosphatase, and phosphorylase kinase gamma 1A suggest a higher glycogen flux in soleus muscle of HSL-null mice. An observed increase in the utilization of glycogen stores supports this finding. Moreover, an increased amount of intramyocellular lipid droplets, observed by transmission electron microscopy, suggests decreased mobilization of lipid stores in HSL-null mice. To complement the transcriptome data, protein expression analysis was performed. Five spots were found to be differentially expressed: pyruvate dehydrogenase E1 alpha, creatine kinase (CK), ankyrin-repeat domain 2, glyceraldehyde-3-phosphate dehydrogenase, and one protein yet to be identified. The increased protein level of CK indicates creatine phosphate degradation to be of increased importance in HSL-null mice. The results of this study suggest that in the absence of HSL, a metabolic switch from reliance on lipid to carbohydrate energy substrates takes place, supporting an important role of HSL in soleus muscle lipid metabolism.
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