Sentez ve mitoz safhalarında senkronize edilmiş a549 akciğer karsinoma hücrelerinde lamin a/c proteininin glikan birimlerinin belirlenmesi
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Dosyalar
Tarih
2020
Yazarlar
Dergi Başlığı
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Cilt Başlığı
Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Nüklear laminler, tip V ara filament proteinleridir ve nukleusa mekanik destek sağlamakla birlikte, kromatin organizasyonu, gen regülasyonu, DNA hasarı ve onarımı, DNA replikasyonu ve sinyal iletimi gibi pek çok hücresel olayda rol oynarlar. Laminlerin en dikkat çekici özelliği ise mitoz bölünme boyunca geriye dönüşümlü olarak ayrılıp birleşmeleridir. Mitoz bölünmenin profaz safhasında laminlerin fosforillenerek ayrıldığı ve geç telofaz safhasında defosforillenerek birleştiği bilinmektedir. Ancak hücre döngüsü boyunca lamin proteinlerinin fosforilasyon dışında bir modifikasyona uğrayıp uğramadığı bilinmemektedir. Bir post-translasyonel modifikasyon olan glikozilasyon, proteinlerin termodinamik, kinetik ve yapısal özelliklerini değiştirerek proteinlerin çevresiyle olan etkileşimini düzenlemektedir. Özellikle nükleositoplazmik proteinlerde O-GlcNAclasyon ve fosforilasyonun proteinin aynı Ser/Thr amino asitleri için yarışmalı olduğu ve bu iki modifikasyonun tersinir olarak aktivite gösterip proteinlerin inaktif veya aktif duruma geçmelerinde rol oynadığı bilinmektedir. Bu bilgilerden hareketle bu tez çalışmasında bu iki modifikasyonun tersinir olarak veya birlikte işlev göstererek hücre döngüsünde lamin A/C proteininin yapısal organizasyonunu düzenliyor olabileceği hipotezi öne sürülmüştür. Bu hipotez doğrultusunda tezin birinci amacı biyoinformatik araçlar kullanılarak insan lamin A/C proteininin olası O-GlcNAc ve O-GalNAc modifikasyonlarını pozisyon spesifik olarak belirlemek ve 3D glikoprotein yapısını oluşturmaktır. İkinci amacı ise hücre döngüsünün G2/M ve S fazlarında senkronize edilmiş A549 akciğer karsinoma hücrelerinde lamin A/C proteininin glikan birimlerini ve fazlar arasındaki glikan değişikliklerini deneysel olarak belirlemektir. Bu amaçla, insan lamin A/C proteininin olası glikozilasyon pozisyonlarının belirlenebilmesi için yüzey alanı, sekonder yapı, hidropati, yüzey ve sıvı erişilebilirliği analizleri gerçekleştirilmiş ve 3D glikoprotein yapısı oluşturulmuştur. Deneysel çalışmalar kapsamında ise hücrelerin doğru bir şekilde senkronize olduğu belirlendikten sonra lektin blotlama ile proteinin uç monosakkaritleri belirlenmiş ve glikozillenmiş lamin A/C proteininin hücredeki lokalizasyonu altın işaretli antikor ve lektinler kullanılarak TEM'de incelenmiştir. Ek olarak, her iki fazda da lamin A/C proteini immünopresipitasyonla saf olarak elde edilip monosakkarit birimleri Cap-LC-ESI/MS-MS ile analiz edilmiştir. Biyoinformatik analizler sonucunda lamin A/C proteininin Ser392'de O-α-GlcNAc, Ser395'de O-β-GlcNAc ve Thr505'de O-GalNAc glikozilasyon modifikasyonları gösterdiği belirlenmiştir. Deneysel veriler göz önüne alındığında, G2/M ve S fazlarında senkronize edilmiş akciğer karsinoma hücrelerinde lamin A/C proteininin her iki fazda da O-GlcNAc, O-GalNAc, galaktoz, mannoz, fukoz ve sialik asit taşıdığı belirlenmiştir. Biyoinformatik analizler sonucunda proteinin N-glikozilasyon pozisyonu taşımadığı belirlendiğinden bu şekerlerin varlığı O-glikozilasyon ile ilişkilendirilmiştir. Bu bulgulara dayanarak lamin A/C'nin O-GlcNAc modifikasyonu ve kor 1 tip O-glikan yapısı üzerinde Sia, Fuc, Man ve O-GalNAc modifikasyonlarını gösteren bir glikoprotein olduğu ortaya konmuştur. TEM çalışmalarıyla ise glikozillenmiş lamin A/C proteininin G2/M fazında nükleoplazmada, S fazında ise nukleus kılıfında ve nükleoplazmada lokalize olduğu gösterilmiştir. Sonuç olarak, bu tez çalışması ile ilk kez hücre döngüsünün interfaz ve mitoz fazlarında lamin A/C proteininin fosforilasyon dışında bir modifikasyon gösterdiği belirlenmiştir. İnterfaz ve mitoz fazlarının her ikisinde de glikozilasyon modifikasyonunun belirlenmesi, lamin A/C proteininin ayrılıp-yeniden birleşmesinde glikozilasyonun, fosforilasyon ile birlikte bir post-translasyonel kod oluşturarak hücre döngüsünün düzenlenmesinde rol aldığını düşündürmektedir. Anahtar sözcükler: Lamin A/C, Glikan, Monosakkarit, A549 hücreleri, İmmünopresipitasyon, CapLC-ESI/MS-MS, TEM
Nuclear lamins are type V intermediate filament proteins that play several roles in many cellular events such as chromatin organization, gene regulation, DNA damage and repair, DNA replication and signal transduction besides providing mechanical support to the nucleus. The most striking feature of the lamins is that their reversible assembly and disassembly during mitosis. In prophase, phosphorylation of lamins triggers the nuclear envelope disassembly and in late telophase, dephosphorylation of lamins reverses the process. However, it hasn't been known whether lamin proteins undergo a modification other than phosphorylation throughout the cell cycle. The post-translational modification, glycosylation, regulates the interaction of proteins with their environment by altering the thermodynamic, kinetic, and structural properties of proteins. It has been known that especially in nucleocytoplasmic proteins, O-GlcNAcylation and phosphorylation are competing for the same Ser/Thr residues of the protein, and the interplay between two modification regulates the activation/inactivation of the protein. Based on this information, in this thesis, we hypothesized that these two modifications may function cooperatively or reversibly to organize the structural organization of the lamin A/C protein during cell cycle. Thus, the first aim of the study is to determine possible O-GlcNAcylated and O-GalNAcylated positions and to generate 3D glycoprotein structure of human lamin A/C protein using bioinformatics tools. The second aim of the study is to analyze experimentally the glycan units of lamin A/C protein and the glycan changes between the phases in A549 lung carcinoma cells synchronized at G2/M and S phases of the cell cycle. For this purpose, in order to determine possible glycosylation positions of human lamin A/C protein, surface area, secondary structure, hydropathy, surface and solvent accessibility analyzes were performed and the 3D glycoprotein structure was generated. Within the scope of the experimental studies, following the synchronization of the cells, the outermost end monosaccharide units of the protein was identified by lectin blotting and glycosylated lamin A/C protein localization in the cell was examined with TEM using colloidal gold conjugated antibody and lectins. In addition, lamin A/C protein was immunoprecipitated and monosaccharide units were analyzed further by Cap-LC-ESI/MS-MS in both two phases. As a result of bioinformatics analysis, lamin A/C protein showed O-α-GlcNAcylation at Ser392, O-β-GlcNAcylation at Ser395, and O-GalNAcylation at Thr505 residues. Considering the experimental data, lamin A/C protein was found to be contain O-GlcNAc, O-GalNAc, galactose, mannose, fucose, and sialic acid in lung carcinoma cells synchronized at G2/M and S phases. Since it was determined with bioinformatics analysis that the protein does not contain N-glycosylation position, the presence of these sugars has been associated with O-glycosylation. Based on these findings, it was demonstrated that lamin A/C is a glycoprotein showing O-GlcNAc modification and Sia, Fuc, Man and O-GalNAc modifications on core 1 type O-glycan structure. TEM studies showed that glycosylated lamin A/C protein is localized in nucleoplasm at G2/M phase and in nuclear envelope and nucleoplasm at S phase. As a result, it is determined for the first time with this thesis that the lamin A/C protein shows a modification other than phosphorylation in interphase and mitosis phases of the cell cycle. The determination of glycosylation modification in both interphase and mitosis suggests that glycosylation plays a role in the regulation of the cell cycle by forming a post-translational code with phosphorylation in assembly and disassembly of the lamin A/C protein during the cell cycle. Keywords: Lamin A/C, Glycan, Monosaccharide, A549 cells, Immunoprecipitation, CapLC-ESI/MS-MS, TEM
Nuclear lamins are type V intermediate filament proteins that play several roles in many cellular events such as chromatin organization, gene regulation, DNA damage and repair, DNA replication and signal transduction besides providing mechanical support to the nucleus. The most striking feature of the lamins is that their reversible assembly and disassembly during mitosis. In prophase, phosphorylation of lamins triggers the nuclear envelope disassembly and in late telophase, dephosphorylation of lamins reverses the process. However, it hasn't been known whether lamin proteins undergo a modification other than phosphorylation throughout the cell cycle. The post-translational modification, glycosylation, regulates the interaction of proteins with their environment by altering the thermodynamic, kinetic, and structural properties of proteins. It has been known that especially in nucleocytoplasmic proteins, O-GlcNAcylation and phosphorylation are competing for the same Ser/Thr residues of the protein, and the interplay between two modification regulates the activation/inactivation of the protein. Based on this information, in this thesis, we hypothesized that these two modifications may function cooperatively or reversibly to organize the structural organization of the lamin A/C protein during cell cycle. Thus, the first aim of the study is to determine possible O-GlcNAcylated and O-GalNAcylated positions and to generate 3D glycoprotein structure of human lamin A/C protein using bioinformatics tools. The second aim of the study is to analyze experimentally the glycan units of lamin A/C protein and the glycan changes between the phases in A549 lung carcinoma cells synchronized at G2/M and S phases of the cell cycle. For this purpose, in order to determine possible glycosylation positions of human lamin A/C protein, surface area, secondary structure, hydropathy, surface and solvent accessibility analyzes were performed and the 3D glycoprotein structure was generated. Within the scope of the experimental studies, following the synchronization of the cells, the outermost end monosaccharide units of the protein was identified by lectin blotting and glycosylated lamin A/C protein localization in the cell was examined with TEM using colloidal gold conjugated antibody and lectins. In addition, lamin A/C protein was immunoprecipitated and monosaccharide units were analyzed further by Cap-LC-ESI/MS-MS in both two phases. As a result of bioinformatics analysis, lamin A/C protein showed O-α-GlcNAcylation at Ser392, O-β-GlcNAcylation at Ser395, and O-GalNAcylation at Thr505 residues. Considering the experimental data, lamin A/C protein was found to be contain O-GlcNAc, O-GalNAc, galactose, mannose, fucose, and sialic acid in lung carcinoma cells synchronized at G2/M and S phases. Since it was determined with bioinformatics analysis that the protein does not contain N-glycosylation position, the presence of these sugars has been associated with O-glycosylation. Based on these findings, it was demonstrated that lamin A/C is a glycoprotein showing O-GlcNAc modification and Sia, Fuc, Man and O-GalNAc modifications on core 1 type O-glycan structure. TEM studies showed that glycosylated lamin A/C protein is localized in nucleoplasm at G2/M phase and in nuclear envelope and nucleoplasm at S phase. As a result, it is determined for the first time with this thesis that the lamin A/C protein shows a modification other than phosphorylation in interphase and mitosis phases of the cell cycle. The determination of glycosylation modification in both interphase and mitosis suggests that glycosylation plays a role in the regulation of the cell cycle by forming a post-translational code with phosphorylation in assembly and disassembly of the lamin A/C protein during the cell cycle. Keywords: Lamin A/C, Glycan, Monosaccharide, A549 cells, Immunoprecipitation, CapLC-ESI/MS-MS, TEM
Açıklama
Anahtar Kelimeler
Lamin A/C, Glikan, Monosakkarit, İmmünopresipitasyon, CapLC-ESI/MS-MS, TEM, A549 Hücreleri, Glycan, Monosaccharide, A549 Cells, Immunoprecipitation, CapLC-ESI/MS-MS, TEM
