Browsing by Subject "Post-translational modification"

Articular cartilage is a highly specialized avascular tissue and consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. The chondrocyte morphology and extracellular matrix properties vary with the depth of cartilage. Some past studies have defined the zonal distribution of a broad range of cartilage proteins in different layers. Based on the variations within each layer, the extracellular matrix can be further distinguished to pericellular, territorial and interterritorial regions. However, most of these studies used guanidine-HCl extraction that leaves an unextracted residual with a substantial amount of collagen. The high abundance of anionic polysaccharide molecules from cartilage adversely affects the chromatographic separation. Scatter oriented chondrocytes only account for the small proportion of the whole tissue protein extraction. However, the density of the cell varies with depth of cartilage as well. Moreover, the physiological status may also altered the extracellular matrix properties. Therefore, a comprehensive strategy to solve all these difficulties are necessary to elucidate the molecular structure of cartilage.

In this study, we used quantitative and qualitative proteomic analysis to investigate various cartilage tissue processing protocols. We established a method for removing chondrocytes from cartilage sections that minimized matrix protein loss. Quantitative and qualitative proteomic analyses were used to evaluate different cartilage extraction methodologies. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. The different extraction methods yielded different protein profiles. For instance, an overwhelming number of collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction.

Subsequently we applied these methods to extract cartilage sections from different cartilage layers (superficial, intermediate and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic). We also utilized lase capture microscopy (LCM) to harvest cartilage sample from individual subregions (territorial and interterritorial regions). The results suggested that there is more unique proteins existed in the superficial layer. By removing the chondrocytes, we were able to identify more extracellular matrix proteins. The phenotyping of cartilage subregions provided the chance to precisely localize the protein distribution, such as clusterin protein. We observed that the guanidine-HCl extractability (guanidine-HCl/ guanidine-HCl + in situ digestion extracts) of cartilage proteins. Proteoglycans showed high extractability while collagen and non-collagenous proteins had lower extractability. We also observed that the extractability might differ with depth of cartilage and also disease states might alter the characters as well.

Laser capture microscopy provides us the access to the cartilage subregions in which only few studies have investigated because of the difficulties to separate them. We established the proteomic analysis compatible-protocol to prepare the cartilage section for LCM application. The results showed that most of the proteoglycans and other proteins were enriched in the interterritorial regions. Type III and VI collagens, and fibrillin-1 were enriched in the territorial regions. We demonstrated that this distribution difference also varied with depth of cartilage. The difference of protein abundance between subregions might be altered because of disease states.

Last we were looking for the post-transliational modification existed in these subregions of cartilage. Deamidation is one of the modification without the enzyme involved. Previous studies have showed that deamidation may accumulated in the tissue with low turnover rate. Our proteomic analysis results suggests that abundance of deamidated peptides also varied in different layers and subregions of cartilage.

We have developed the monoclonal antibody based immunoassay to quantify the deamidated cartilage oligomeric matrix protein within cartilage tissue from different joints (hip and knee) and disease states (healthy, para-lesion, and remote lesion). The results suggests that the highest concentration of deamidated COMP was identified in arthritic hip cartilage.

The results of this study generated several reliable protocols to perform cartilage matrix proteomic analysis and provided data on the cartilage matrix proteome, without confounding by intracellular proteins and an overwhelming abundance of collagens. The discovery results elucidated the molecular architecture of cartilage tissue at different joint sites and disease states. The similarities among these cartilages suggested a constitutive role of some proteins such as collagen, prolargin, biglycan and decorin. Differences in abundance or distribution patterns, for other proteins such as for cartilage oligomaric matrix protein, aggrecan and hyaluronan and proteoglycan link protein, point to intriguing biological difference by joint site and disease state. Decellularization and a combination of extraction methodologies provides a holistic approach in characterizing the cartilage extracellular matrix. Guanidine-HCl extractability is an important marker to characterize the statue of cartilage; however it has not been fully understand. The protein distributions in matrix subregions may also serve as an index to characterize the metabolic status of cartilage in different disease states. A large sample cohort will be necessary to elucidate these characters.

Eukaryotic Topoisomerase 2 is an essential enzyme that solves DNA topological problems such as DNA knotting, catenation, and supercoiling. It alters the DNA topology by introducing transient double strand break in one DNA duplex as a gate for the passage of another DNA duplex. Two different aspects of studies about eukaryotic Topoisomerase 2 will be covered in this thesis. In the first half of the thesis, we investigated conformational changes of human Topoisomerase 2 (hsTop2) in the presence of cofactors and inhibitors. In the second half, we focused on an unknown regulatory function in the C-terminal domain (CTD) of Drosophila Topoisomerase 2 (Top2).

In the project of studying enzyme conformational changes, we adapted a previously developed methodology, Pulse-Alkylation Mass Spectrometry, with monobromobimane to study the protein dynamics of hsTop2. Using this method, we captured the evidence of conformational changes in the presence of ATP and Mg2+ or the Top2 inhibitor, ICRF-193 which were not previously observed. Last, by using CTD truncated hsTop2, the increasing reactivity of Cys427 suggested the CTD domain might be tethered adjacent to the core enzyme.

Following the study of enzyme conformational changes, we switched gear to examine an interaction between Drosophila Top2 and Mus101, homolog of human TopBP1. We first found that Mus101 interacts with CTD of Top2 in a phosphorylation-dependent manner. Next, in the co-immunoprecipitation and pull-down experiments using truncated or mutant Top2 with various Ser to Ala substitutions, we mapped the binding motif to the last amino acids of Top2 and identified that phosphorylation of Ser1428 and Ser1443 is important for Top2 to interact with the N-terminus of Mus101, which contains BRCT1/2 domains (BRCT, BRCA1 C-terminus). The binding affinity of the N-terminal Mus101 with a synthetic phosphorylated peptide covering the last 25 amino acids of Top2 (with pS1428 and pS1443) was determined by surface plasmon resonance with a Kd of 0.57 μM. In an in vitro decatenation assay, Mus101 can specifically reduce the decatenation activity of Top2, and dephosphorylation of Top2 attenuates this response to Mus101. Next, we endeavored to establish a cellular system for testing the biological function of Top2-Mus101 interaction. Top2-silenced S2 cells rescued by Top220, truncation of 20 amino acids from the C-terminus of Top2, developed abnormally high chromosome numbers, which implies an infidelity in chromosome segregation during mitosis. Lastly, Top2-null flies rescued by Top2 with S1428A and S1443A were found to be viable but sterile. After investigating spermatogenesis, telophase of meiosis I was delayed, indicating Top2-Mus101 interaction is also important in segregating DNA in meiosis.