Cells respond to their environment through dynamic posttranslational modification of their existing proteins. There are more than 20 posttranslational modifications that occur on eukaryotic proteins. Several of these modifications, with phosphorylation being the hallmark, participate in signal transduction. Generally, glycosylation is not thought to participate directly in signaling. Complex N-and 0-linked glycosylation occurs on membrane-bound or secreted proteins that are synthesized in the endoplasmic reticulum and Golgi apparatus. The lumenal or extracellular localization ofthese glycans restricts their potential for dynamic responsiveness to signals. In contrast, 0-GlcNAc is a simple monosaccharide modification that is abundant on serine or threonine residues ofnucleocytoplasmic proteins. An 0-GlcNAc site consensus motif has not yet been identified. However, many attachment sites are identical to those used by serine/threonine) kinases, and a neural network program has been developed to predict 0GlcNAc sites. The dynamic glycosylation of serine or threonine residues on nuclear and cytosolic proteins by 0-linked beta-N-acetylglucosamine (0-GlcNAc) is abundant in all multicellular eukaryotes. On several proteins, 0-GlcNAc and 0-phosphate alternatively occupy the same or adjacent sites, leading to the hypothesis that one function of this saccharide is to transiently block phosphorylation. Many proteins have been identified that carry this modification, including transcription factors, cytoskeletal proteins, nuclear pore proteins, oncogene products, and tumor suppressors. 0-GlcNAc appears to modify a large number of nucleocytoplasmic proteins· One of important regulatory proteins on which this project concentrates is β-catenin. Here, we examined where does this type ofposttranslational modification takes place on the protein. Our results indicated that P-catenin is 0-glycosylated on both the N-terminus and Cterminus, but not at the ARMADILLO segment. Further, we show that the known phosphorylation sites located at theN-terminal "destruction box" of this protein are not involved in 0-glycosylation. Furthermore, we demonstrated that the threonines adjacent to phosphorylation-site Threonin41 are not essential in 0-glycosylation process. In addition, treatment ofprostate cancer lines with PUGNAc, a non-cytotoxic reversible inhibitor ofOGlcNAcase, caused a decrease in the expression oftransfected P-catenin in the nucleus with increasing cellular 0-glycosylation ofthe protein suggesting that 0-glycosylation was hindering P-catenin's nuclear translocation. Additional studies showed that 0-glycosylation of P-catenin decreased transcriptional activity of a TopFlash reporter plasmid. In summary, our results show that P-catenin is 0-glycosylated on theN-and C-terminus, but not on ARMADILLO segment, and that phosphorylation sites are not the critical for 0-glycosylation. Furthermore, our data show that 0-glycosylation of P-catenin may represent a novel mechanism important in the regulation of the nuclear localization and transcriptional activity of P-catenin. / Thesis / Master of Science (MSc)

Converging evidence indicates that SOD1 aggregation is a common feature of mutant SOD1 (mSOD1)-linked FALS, and seems to be directly related to the gain-of-function toxic property. However, the mechanisms of protein aggregation are not fully understood. To study the contribution of modification on cysteine residues in SOD1 aggregation, we systematically examined the redox state of SOD1 cysteine residues in the G37R transgenic mouse at different stages of ALS and under oxidative stress induced by H2O2. Our data showed that under normal circumstances, cysteine 111 in SOD1 is free. Under oxidative stress, it is prone to oxidative modification by providing the thiolate anion (S-). With the progression of ALS, increased levels of oxidative insults facilitated the oxidation of thiol groups of cysteine residues. Human mutant SOD1 could generate an upper shifted band in SDS-PAGE, which turned out to be a Cys111-peroxidized SOD1 species. We also found that at different stages of ALS, accumulated oxidative stress facilitated the aggregates formation, which were not mediated by disulfide bond. The oxidative modification of cysteine 111 may promote the formation of disulfide bond-independent SOD1 aggregates. In addition, we investigated the correlation between nitrosative stress and S-nitrosylation of protein disulfide isomerase (PDI) in the mechanism of aggregates formation. Our data showed that up-regulated inducible nitric oxide synthase (iNOS) generated high levels of nitric oxide (NO), which induced S-nitrosylation of PDI with the progression of ALS in the spinal cords of mSOD1 transgenic mice. This correlation was confirmed by treating SH-SY5Y cells with NO donor SNOC to trigger the formation of S-nitrosylated PDI (SNO-PDI). When mSOD1 was overexpressed in SH-SY5Y cells, iNOS expression was up-regulated, NO generation was increased consequently. Furthermore, both SNO-PDI and mSOD1 aggregates were detected in these cells. Blocking NO generation with NOS inhibitor N-nitro-L-arginine (NNA) attenuated the S-nitrosylation of PDI; the formation of mSOD1 aggregates was inhibited as well. We conclude that NO-mediated S-nitrosylation of PDI is highly linked to the accumulation of mSOD1 aggregates in ALS.

Converging evidence indicates that SOD1 aggregation is a common feature of mutant SOD1 (mSOD1)-linked FALS, and seems to be directly related to the gain-of-function toxic property. However, the mechanisms of protein aggregation are not fully understood. To study the contribution of modification on cysteine residues in SOD1 aggregation, we systematically examined the redox state of SOD1 cysteine residues in the G37R transgenic mouse at different stages of ALS and under oxidative stress induced by H2O2. Our data showed that under normal circumstances, cysteine 111 in SOD1 is free. Under oxidative stress, it is prone to oxidative modification by providing the thiolate anion (S-). With the progression of ALS, increased levels of oxidative insults facilitated the oxidation of thiol groups of cysteine residues. Human mutant SOD1 could generate an upper shifted band in SDS-PAGE, which turned out to be a Cys111-peroxidized SOD1 species. We also found that at different stages of ALS, accumulated oxidative stress facilitated the aggregates formation, which were not mediated by disulfide bond. The oxidative modification of cysteine 111 may promote the formation of disulfide bond-independent SOD1 aggregates. In addition, we investigated the correlation between nitrosative stress and S-nitrosylation of protein disulfide isomerase (PDI) in the mechanism of aggregates formation. Our data showed that up-regulated inducible nitric oxide synthase (iNOS) generated high levels of nitric oxide (NO), which induced S-nitrosylation of PDI with the progression of ALS in the spinal cords of mSOD1 transgenic mice. This correlation was confirmed by treating SH-SY5Y cells with NO donor SNOC to trigger the formation of S-nitrosylated PDI (SNO-PDI). When mSOD1 was overexpressed in SH-SY5Y cells, iNOS expression was up-regulated, NO generation was increased consequently. Furthermore, both SNO-PDI and mSOD1 aggregates were detected in these cells. Blocking NO generation with NOS inhibitor N-nitro-L-arginine (NNA) attenuated the S-nitrosylation of PDI; the formation of mSOD1 aggregates was inhibited as well. We conclude that NO-mediated S-nitrosylation of PDI is highly linked to the accumulation of mSOD1 aggregates in ALS.

Rapid and well-orchestrated action potential propagation through the myocardium is essential to each heartbeat. Gap junctions comprising primarily Cx43 reside within the intercalated discs connecting cardiomyocytes, effecting not only direct intercellular electrical coupling, but the localization of other junctional structures and ion channels. Alterations in Cx43 expression occur in essentially all forms of heart disease and is therefore a topic of intense study. Posttranslational modification of Cx43 is understood to impact trafficking, conduction, and stability. Altered Cx43 phosphorylation is well described during pathological remodeling of gap junctions in response to cellular stress. Research has revealed how phosphorylation of specific residues elicit specific effects on Cx43, but the complexity of this process has left much unknown. In particular, the role phosphorylation of a triplet of double serine residues, Ser365, Ser368, and Ser373, plays in GJ function and Cx43/14-3-3 interaction has been called into question. Using an ex vivo whole heart ischemia model we find a decrease in pS368 in mice lacking the ability to phosphorylate S365 and S373 while under stress. In vitro transfection of human induced pluripotent stem cell-derived cardiomyocytes when stressed with PMA were also carried out. These data allow us to piece together the exquisite interplay of gatekeeper phosphorylation events upstream of channel closure, altered protein-protein interactions, and gap junction internalization and degradation. It is hoped that our increasing understanding of this important area of gap junction biology will facilitate better understanding of arrhythmogenesis, and potential therapeutic strategies to restore or preserve normal electrical coupling in diseased hearts. / Master of Science / The heart, an electrically active organ, relies on the propagation of an electrical signal throughout its entirety in order to produce a healthy heartbeat. In order to do so, the heart uses specialized muscle cells known as cardiomyocytes which can not only contract but pass along chemical signals to the cardiomyocyte next in line to signal it to contract as well. The passage of signals occurs through protein units called gap junctions and are made predominantly of Cx43 proteins in the heart. Gap junctions look and function like tubes that travel from the inside space of one cell to the other and allow a flow of small molecules to occur; these small molecules, namely ions, are part of the signal needed to initiate contraction in the adjacent cell. Cx43, like many proteins in our bodies, is slightly altered after it is produced through a process known as posttranslational modification. This allows the cell to alter the localization and function of the protein and tailor it for the needs of the cell. Rather than changing the backbone composition of the protein, small chemical groups are attached, and this imparts a change to how the protein interacts with other proteins or its environment. In particular, one form of modification is known as phosphorylation where a phosphate group is attached to the protein at specific locations along its chain. Cx43 too can be phosphorylated, and while under pathological stress, such as a lack of oxygen or infection, cardiomyocytes increase the amount of phosphorylated Cx43 at a site known to cause pathological changes to the function of Cx43. These changes include how well the gap junctions can transmit signals or associate with other proteins and, in the heart, can predispose the development of arrhythmias or unhealthy heartbeats. However, not all phosphorylation is bad and phosphorylation at other locations also occurs during normal healthy functions of the cardiomyocyte can affect how other sites along Cx43 are phosphorylated. The process of one phosphorylated site affecting another is known as the gatekeeper effect and add a new layer to our understanding of how cells use phosphorylated Cx43 to fine tune its effects. Using cells that do not produce their own Cx43 and subsequently giving them the instructions to produce specific forms of mutant Cx43 that can and cannot be phosphorylated at specific sites, we can understand with greater detail of how cardiomyocytes respond to stress and how some of those responses can be pathological. This will allow future research into the creation of therapies that prevent negative Cx43 phosphorylation after illness, potentially avoiding the development of dangerous arrhythmias.

<p>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. </p><p>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. </p><p>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. </p><p>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. </p><p>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. </p><p>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. </p><p>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.</p> / Dissertation

Protein phosphorylation is a very common posttranslational modification (PTM), which lately has been found to hold the keyrole in the development of many severe diseases, including cancer. Thereby, phosphoprotein analysis tools, generally based on specific enrichment of the phosphoryl group, have been a hot topic during the last decade. In this thesis, two new TiO2-based on-target enrichment methods are developed and presented together with enlightening fundamental results. Evaluation of the developed methods was performed by the analysis of: custom peptides, β-casein, drinking milk, and the viral protein pIIIa. The results show that: i) by optimizing the enrichment protocol (first method), new phosphorylated peptides can be found and ii) by the addition of a separation step after the enrichment (second method), more multi-phosphorylated peptides, which usually are hard to find, could be detected. The fundamental part, on the other hand, shows that the phosphopeptide adsorption is caused by electrostatic interactions, in general follows the Langmuir model, and the affinity increases with the phosphorylation degree. Here, however, the complexity of the system was also discovered, as the adsorption mechanism was found to be affected by the amino acid sequence of the phosphopeptide.

The class II Transactivator (CIITA) is the master regulator for Major Histocompatibility Class II (MHC II) molecules. CIITA is dynamically regulated by a series of Posttranslational Modifications (PTMs). CIITA is responsible for initiating transcription of MHC II genes, thus allowing peptides derived from extracellular antigens to be presented to CD4+ T cells. CIITA’s PTMs are necessary for regulation of CIITA’s location, activity, and stability. Our work identifies the kinase complex ERK1/2 as being responsible for phosphorylating the previously identified regulatory site, serine (S) 280 on CIITA. Phosphorylation by ERK1/2 of CIITA S280 leads to increased levels of CIITA mono-ubiquitination and overall increases in MHC II activity. We further identify a novel ubiquitin modification on CIITA, lysine (K) 63 linked ubiquitination poly ubiquitination. Our data shows novel crosstalk between K63 ubiquitination and ERK1/2 phosphorylation. K63 ubiquitinated CIITA is concentrated to the cytoplasm, and upon phosphorylation by ERK1/2, CIITA translocates to the nucleus, thus demonstrating that CIITA’s location and activity is regulated through PTM crosstalk. While ubiquitination has been shown to be a critical PTM in the regulation of CIITA, the enzyme(s) mediating this important modification remained to be elucidated. Previous reports implicating the histone acetyltransferase (HAT), pCAF as an ubiquitin E3 ligase were intriguing, as pCAF is also known to participate in the acetylation of both histones at the MHC II promoter and in acetylation of CIITA. We now identify novel roles for pCAF in the regulation of CIITA. We show pCAF acts as an E3 ligase, mediating mono, K63, and K48 linked ubiquitination of CIITA. We therefore demonstrate an additional substrate for the “dual acting” enzyme, pCAF. In sum, our observations identify enzymes involved in both the phosphorylation and ubiquitination of key residues of CIITA, which ultimately regulate CIITA activity. Together our observations contribute to knowledge of CIITA’s growing network of PTMs and their role in regulating the adaptive immune response, and will allow for development of novel therapies to target dysregulated CIITA activity during adaptive immune responses.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 5 uppsatser.

Angiogenesis is the process of new blood vessel growth from preexisting vessels. This process relies on the activity of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) and occurs in both normal and pathological tissues. Angiogenesis is often dysregulated in diseases such as cancer and many efforts have been made to treat such diseases by targeting the VEGFR-2 pathway. VEGFR-2 is activated upon ligand binding and subsequent autophosphorylation of tyrosine residues in the kinase domain, which leads to endothelial cell survival, proliferation, and growth – all of which are required for angiogenesis to occur. It was previously demonstrated that methylation of VEGFR-2 at Lys1041 enhanced its tyrosine autophosphorylation and is required for VEGFR-2 mediated angiogenesis in zebrafish and tumor growth in mouse. However, the Lysine Methyltransferase (KMT) involved in the methylation of VEGFR-2 remains unknown. This study aimed to identify the KMT involved in the methylation of VEGFR-2. We have identified Enhancer of zeste homolog 2 (EZH2) as the KMT likely responsible for catalyzing methylation of K1041 on VEGFR-2. Over-expression of EZH2 was found to increase phosphorylation of Tyr1054, one of the required phosphorylation sites for VEGFR-2 activation, in whole cell lysates and VEGFR-2 purified by immunoprecipitation. The effect of over-expression of EZH2 in the phosphorylation of VEGFR-2 at Tyr1054 was dose-dependent - increasing concentrations of EZH2 resulted in increasing phosphorylation of VEGFR-2 at Tyr1054. Moreover, we determined that EZH2 physically interacts with VEGFR-2 as demonstrated by co-immunoprecipitation in vitro GST-pulldown assays. The C-terminus of EZH2 (amino acids 371-746), physically interacted with VEGFR-2. Taken together, we have identified EZH2 as a candidate KMT involved in the methylation of Lys1041, which increases phosphorylation of VEGFR-2 at Tyr1054. / 2020-07-03T00:00:00Z

No description available.