TR Dizin İndeksli Yayınlar / TR Dizin Indexed Publications Collection

Permanent URI for this collectionhttps://hdl.handle.net/11147/7149

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2017 - 20192

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Department: search.filters.department.İzmir Institute of Technology. Molecular Biology and GeneticsPublisher: search.filters.publisher.Türk Biyokimya Derneği

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  • Article
    Citation - WoS: 2
    Citation - Scopus: 2
    Identification of Cytoplasmic Sialidase Neu2-Associated Proteins by Lc-ms/Ms
    (Türk Biyokimya Derneği, 2019) Akyıldız Demir, Seçil; Seyrantepe, Volkan
    Background: Cytoplasmic sialidase (NEU2) plays an active role in removing sialic acids from oligosaccharides, gly-copeptides, and gangliosides in mammalian cells. NEU2 is involved in various cellular events, including cancer metabolism, neuronal and myoblast differentiation, proliferation, and hypertrophy. However, NEU2-interacting protein(s) within the cell have not been identified yet. Objective: The aim of this study is to investigate NEU2 interacting proteins using two-step affinity purification (TAP) strategy combined with mass spectrometry analysis. Methods: In this study, NEU2 gene was cloned into the pCTAP expression vector and transiently transfected to COS-7 cells by using PEI. The most efficient expression time of NEU2- tag protein was determined by real-time PCR and Western blot analysis. NEU2-interacting protein(s) were investigated by using TAP strategy combined with two different mass spectrometry experiment; LC-MS/MS and MALDI TOF/TOF. Results: Here, mass spectrometry analysis showed four proteins; a-actin, beta-actin, calmodulin and histone H1.2 proteins are associated with NEU2. The interactions between NEU2 and actin filaments were verified by Western blot analysis and immunofluorescence analysis. Conclusions: Our study suggests that association of NEU2 with actin filaments and other protein(s) could be important for understanding the biological role of NEU2 in mammalian cells.
  • Article
    Alteration of Protein Localization and Intracellular Calcium Content Due To Connexin26 D50a and A88v Mutations
    (Türk Biyokimya Derneği, 2017) Aypek, Hande; Meşe, Gülistan
    Introduction: Connexins (Cx) play essential roles in cellular homeostasis by forming gap junctions and non-junctional hemichannels. In vitro characterization of Cx26 mutations causing keratitis-ichthyosis-deafness (KID) syndrome, were shown to form leaky hemichannels. The molecular/ cellular mechanisms affected by aberrant hemichannels have recently been elucidated. Here, we further wanted to characterize Cx26 KID syndrome mutations, D50A and A88V, which were shown to form aberrant hemichannels and remained unaddressed in the literature. Methods: Neurobiotin uptake assay in HeLa and N2A cells transfected with Cx26-WT, D50A or A88V verified the presence of aberrant hemichannels and immunofluorescent staining with fluorescent microscopy determined cellular localization of Cx26. Finally, intracellular calcium content was examined by using calcium indicator, Fluo-3AM, and flow cytometer. Results: Cx26-D50A and A88V mutations prevented the formation of gap junction plaques at cell-cell appositions and mutant proteins were observed to localize to the Golgi apparatus. Further, comparison of intracellular calcium content showed an increase in calcium amount in cells containing Cx26-D50A and A88V relative to Cx26-WT. Conclusion: Retention of Cx26 in the Golgi apparatus and alteration in the intracellular calcium content due to KID syndrome mutations may influence various cellular processes that might contribute to development of epidermal phenotypes.