Identification Effects Of Protein Kinase C Activation In Neurons Under Physiological And Degenerative Conditions

Tez (Doktora) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2012Thesis (PhD) -- İstanbul Technical University, Institute of Science and Technology, 2012Nöronlar ileri derecede farklılaşmış hücreler olup normal koşullar altında hücre döngüsünün G0 evresinde yer alırlar. Ancak, Alzheimer hastalığı (AH) gibi nörodejeneratif durumlarda nöronların G0 evresini terk ettiği, G1/S kontrol noktasını atlayarak hücre döngüsüne devam ettiği, hatta DNA’sını replike ettiği gösterilmiştir. AH’nda sinyal iletim yolaklarının birçoğu değişime uğramaktadır. Ser/Thr protein kinaz ailesinin bir üyesi olan Protein Kinaz C (PKC) de AH’nda ekspresyon ve aktivasyon düzeyleri değişime uğrayan proteinler arasında yer almaktadır. PKC’nin aktivasyonu genellikle bu proteinin diaçilgliserol, araşidonik asit gibi ikincil mesajcı sinyal moleküllerince hücre membran kompartmanlarına lokalizasyonu ile sağlanır. Bununla birlikte, PKC’ye nükleusta meydana gelen olaylarda görevler atfeden bulgular ve görüşler de artmaktadır. Kısacası PKC’nin hücre içi konumu ve görevleri özellikle nöronlar için net olarak tanımlanamamıştır. PKC, nörodejenerasyon ile ilişkili yolaklardaki görevinin yanı sıra, hücre bölünmesi ve hatta hücre farklılaşması gibi olaylar ile de ilişkilendirilmektedir. Tüm bu veriler ışığında, bu çalışmanın amacı nöronlarda fizyolojik ve dejeneratif koşullar altında Phorbol 12-myristate 13-acetate (PMA) kaynaklı PKC aktivasyonunu etkilerini araştırmaktır. Bu kapsamda nöronlarda PKC ve siklinlerin ekspresyon düzey ve lokalizasyon farklılıkları ile nöronal sitoiskelet yapısındaki değişimler araştırılmıştır. Bu çalışma nükleer PKC’nin hücre siklusuyla ilişkili hali hazırda bilinen görevlerine ilaveten nöronlarda nükleer siklinD1 artışı ve mikrotubullerin yeniden organizasyonu yardımı ile farklılaşmada gerilemeye yol açtığını göstermesi bakımından son derece önemlidir.Neurons are highly differentiated cells that reside in G0 phase of the cell cycle under normal circumstances. However, in neurodegenerative conditions such as Alzheimer’s disease (AD) it was shown that neurons leave G0 phase, bypass G1/S checkpoint and continue cell cycle and even replicate their DNAs. Several alterations of many signal transduction pathways in AD have been reported and a member of Ser/Thr kinase family proteins, Protein Kinase C (PKC) is one of these proteins that have altered expression and activation levels in AD. Activation of PKC generally depends on its targeting to the membrane compartments by second messengers such as diacylglycerol and arachidonic acid. However, there is also a growing body of evidence attributing nuclear functions for PKC. Hence, its precise subcellular distribution and roles are still unclear in neurons. Besides its role in signaling pathways which are associated with neurodegeneration, PKC has also been associated with proliferation and even differentiation. In this content the purpose of this study was to investigate the effects of PKC activation by Phorbol 12-myristate 13-acetate (PMA) in physiological and degenerative neurons. For this purpose, expressional changes and distribution of PKC and cyclins in hippocampal neurons were analyzed. This study is very important as it showed that nuclear PKC, in addition to its previously known roles in cell cycle regulation, caused withdrawal from differentiation in neurons by increasing nuclear cyclinD1 and promoting microtubule re-organization.DoktoraPh

Regulation Of Microtubule Severing

Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2006Mikrotübüller, alfa ve beta tubulin heterodimerlerinden oluşan hücre iskeleti polimerleridirler. Hücre içi taşıma, hücre hareketi, hücre bölünmesi gibi olaylarda görevlidirler. Sinir hücrelerinde, MTler hücrenin büyümesine destek sağlamakla beraber hücresel yapının şekillenmesinde, akson ve dendritlerin oluşumunda da etkilidirler. Katanin ve spastin MT kesici enzimlerdir. Katanin iki alt üniteden oluşur, p60 ve p80. Spastin, p60–katanin alt ünitesi ile büyük benzerlik göstermektedir. Spastinin en fazla ilgi çeken proteinlerden biri haline gelmesinin nedeni de bu proteinin MT temel hücre biyolojisini nörolojik hastalıklarla birleştirmesidir. Spastinin mutasyonunda kalıtsal spastik parapleji rahatsızlığına neden olduğu bilinmektedir. Çalışmadaki hedefimiz spastinin karakterizasyonunda yeni adımlar atabilmek, MTlerin spastin, katanin tarafından kesilmesinin mikrotübül ilişkili proteinler tarafından düzenlenmesini aydınlatmaya çalışmaktır. Çalışmanın bu kısmında MTler üzerinde koruyucu görevleri olan mikrotübül–ilişkili proteinler, özellikle de MAP1b ve MAP2c üzerinde durulmuştur. Spastinin görevini aydınlatabilmek için hipokampüs hücrelerinde GFP, spastin, p60–katanin proteinlerinin ekspresyonu gerçekleştirildi. Hücreler transfeksiyonu takip eden 2. ve 4. günlerde sabitlendi. Birincil ve ikincil antikorlar ile boyandıktan sonra hücreler flüoresan mikroskobu ile incelendi. Kontrol hücreleri, katanin ve spastin proteinlerini aşırı eksprese eden hücreler aralarında karşılaştırıldı. Çalışmanın ikinci kısmında ise mikrotübül ilişkili proteinlerin, p60–katanin ve spastin eksprese eden hücreler üzerinde herhangi bir düzenleyici rolünün olup olmadığı aydınlatılmaya çalışıldı. P60–katanin ve spastin proteinleri mikrotübül ilişkili proteinlerin varlığında ya da bu proteinler olmadan fibroblast hücrelerinde eksprese edildiler. Transfeksiyonu takip eden günde hücreler sabitlendi. Birincil ve ikincil antikorlarla işaretlendikten sonra hücreler flüoresan mikroskobunda incelendiler.Microtubules are cytoskeletal polymers made of alpha and beta tubulin heterodimers. They have role in cell transport, cell motility and cell division. In neurons, MTs provide support for the growth and maintenance of the axonal and dendritic processes. Katanin and spastin are MT severing enzymes. Katanin has two subunits, p60 and p80. P60 subunit has high homology with spastin. Spastin became popular because it has brought basic MT cell biology into the neurological disorders. It is known that spastin mutation leads to neurological disorder, hereditary spastic paraplegia. Our aim was to characterize spastin and also to identify the regulation mechanisms of MT severing by spastin and katanin. We concentrated on MAP1b and MAP2c proteins that have protective role over MTs. In the first part of the study, we have worked with hippocampus cells. We overexpressed GFP, p60 –katanin and spastin constructs in hippocampal cells. Cells were fixed at the particular time points following transfection. After fixation, immunostaining was done and then by using primary and secondary antibodies cells were analyzed with fluorescent microscopy. Control cells, p60 –katanin overexpressing and spastin overexpressing cells were compared with each others. In the second part of the work, MAP’s protective functions were analyzed in spastin and p60–katanin over expressing cells. P60 –katanin and spastin constructs were overexpressed with and without MAP1b and MAP2c in living fibroblast cells, RFL-6. Cells were fixed on the following day of transfection and stained with primary and secondary antibodies. After immunostaining, cells were analyzed with fluorescent microscopy.Yüksek LisansM.Sc

p53: Transcription factor - regulatory partner for p60-katanin

Conference: 2nd International Congress of the molecular Biology Association of Turkey 22-23 November 2013,İstanbul,Turkey (Poster Sunum)#nofulltext#

p60-katanin: a novel interacting partner for p53

Katanin, one of the best-characterized microtubule (MT) severing proteins, is composed of two subunits: catalytic p60-katanin, and regulatory p80-katanin. p60-katanin triggers MT reorganization by severing them. MT reorganization is essential for both mitotic cells and post-mitotic neurons in numerous vital processes such as intracellular transport, mitosis, cellular differentiation and apoptosis. Due to the deleterious effect of continuous severing for cells, p60-katanin requires a strategic regulation. However, there are only a few known regulators of p60-katanin. p53 functions in similar cellular processes as katanin such as cell cycle, differentiation, and apoptosis depending on its interacting partners. Considering this similarity, in this study we investigated p53 as a potential regulatory candidate of p60-katanin, and examined their interaction. Co-immunoprecipitation analyses revealed that p60-katanin interacts with p53. We were able to locate a potential interaction site for the two proteins by deleting different candidate regions We showed for the first time that p53 and p60-katanin interact. This interaction appears to occur via p53's DNA binding domain and p60-katanin's C-terminal. This study will pave the way for future studies regarding the functional outcomes of this interaction which is vital for understanding the regulation of cellular events such as cell cycle, differentiation, and apoptosis in disease and in health

Neuroprotective strategies against calpain-mediated neurodegeneration

WOS: 000348987000001PubMed ID: 25709452Calpains are calcium-dependent proteolytic enzymes that have deleterious effects on neurons upon their pathological over-activation. According to the results of numerous studies to date, there is no doubt that abnormal calpain activation triggers activation and progression of apoptotic processes in neurodegeneration, leading to neuronal death. Thus, it is very crucial to unravel all the aspects of calpain-mediated neurodegeneration in order to protect neurons through eliminating or at least minimizing its lethal effects. Protecting neurons against calpain-activated apoptosis basically requires developing effective, reliable, and most importantly, therapeutically applicable approaches to succeed. From this aspect, the most significant studies focusing on preventing calpain-mediated neurodegeneration include blocking the N-methyl-D-aspartate (NMDA)-type glutamate receptor activities, which are closely related to calpain activation; directly inhibiting calpain itself via intrinsic or synthetic calpain inhibitors, or inhibiting its downstream processes; and utilizing the neuroprotectant steroid hormone estrogen and its receptors. In this review, the most remarkable neuroprotective strategies for calpain-mediated neurodegeneration are categorized and summarized with respect to their advantages and disadvantages over one another, in terms of their efficiency and applicability as a therapeutic regimen in the treatment of neurodegenerative diseases.Turkish Academy of Sciences Distinguished Young Scientist Award (TUBA-GEBIP); Scientific and Technological Research Council of Turkey (TUBITAK)-The Basic Sciences Research Group (TBAG) [108T811]The "Speedy/RINGO over-expression to prevent calpain-mediated apoptosis" study was funded by grants to Arzu Karabay from The Turkish Academy of Sciences Distinguished Young Scientist Award (TUBA-GEBIP) and The Scientific and Technological Research Council of Turkey (TUBITAK)-The Basic Sciences Research Group (TBAG) (grant number 108T811)

HNF1A-MODY Mutations in Nuclear Localization Signal Impair HNF1A-Import Receptor KPNA6 Interactions

#nofulltext# --- Fareed, Fareed M. A. (Arel Author), Çapan Yalçın, Özlem (Arel Author)Mutations in hepatocyte nuclear factor (HNF)1A gene cause the most common form of Maturity-onset diabetes of the young (MODY), a monogenic subtype of diabetes mellitus. Functional characterization of mutant proteins reveals that mutations may disrupt DNA binding capacity, transactivation ability and nuclear localization of HNF1A depending on the position of the mutation. Previously identified Arg271Trp and Ser345Tyr mutations in HNF1A were found to be defective in nuclear localization. Arg271 residue resides in a region similar to classical nuclear localization signal (NLS) motif, while Ser345 does not. Importin alpha family members recognize NLS motifs on cargo proteins and subsequently translocate them into nucleus. Here, we first investigated the nuclear localization mechanism of wild type HNF1A protein. For this purpose, we analyzed the interaction of HNF1A with three mouse homolog importin alpha proteins (KPNA2, KPNA4 and KPNA6) by co-immunoprecipitation assay and molecular docking simulation. Hereby, KPNA6 was identified as the main import receptor, which is responsible for the transport of HNF1A into the nucleus. Immunolocalization studies in mouse pancreatic cells (Min6) also confirmed the co-localization of HNF1A and KPNA6 in the cytoplasm. Secondly, the interaction between KPNA6 and mutant HNF1A proteins (Arg271Trp and Ser345Tyr) was assessed. Co-immunoprecipitation studies revealed a reduced interaction compared to wild type HNF1A. Our study demonstrated for the first time that HNF1A transcription factor is recognized and transported by importin/karyopherin import family, and mutations in NLS motifs may disrupt the interaction leading to nuclear localization abnormalities and MODY phenotype