Moleküler Biyoloji ve Genetik

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Importance of ASC expression for melanoma tumorigenesis in vivo and a role for inflammasome activation in apoptosis induction
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2010., 2010.) Çifdalöz, Metehan.; Özören, Nesrin.
ASC is an adaptor protein which is capable of binding to numerous partners due to its two oligomerization domains, PYRIN and CARD. Its involvement in the inflammasome complex as an essential adaptor, in the regulation of the cleavage and secretion of IL-1β and IL-18, in apoptosis induction and its evident silencing via methylation in variety of cancer types make ASC an important protein to be examined. Preliminary data from our laboratory showed that introduction of ASC into melanoma cell lines, with silenced ASC, via lentiviral transfection decreased the number and the size of the colonies formed by these cells in an anchorage independent medium up to 50 per cent. In this study, first, we hypothesized that injection of ASC-introduced human melanoma cell lines into immunodeficient mice may show the importance of ASC silencing in human melanoma progression. We observed a slight difference in the tumorigenicity between ASC expressing and control groups. Secondly, we tested the importance of ASC expression in tumorigenesis using mice with a normal immune system. In these experiments, mouse melanoma cells with varying ASC expression levels were utilized. We also transduced the ASC non-expression mouse melanoma cell line (B16 F1) with the mouse ASC gene. Unfortunately, with the small number of animals, we could not observe a clear reduction in the tumor sizes produced by these cells in an ASC dependent manner. Finally, we aimed to analyze the combinatorial/synergistic effect of the DNA damaging agent (etoposide) and an inflammasome activating substrate (imiquimod) treatment on the chemoresistance of ASC-introduced and control melanoma cell lines. We observed a combinatorial effect of treatment with both agents in SKMEL-19 and SKMEL- 28 cell lines, although this effect was regardless of the ASC expression status.
Importance of ASC expression levels in the development of chemoresistance in human melanoma cell lines
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2008., 2008.) Erdoğan, Damla.; Özören, Nesrin.
ASC (Apoptosis-associated speck like protein containing a CARD) is an adapter protein containing PYRIN and CARD interaction domains. ASC is implicated in apoptosis and innate immune signaling and is required for IL-1 and IL-18 processing via the activation of caspase 1. ASC protein expression is suppressed via methylation of its promoter regions in a high percentage of melanoma and other cancer types, including colorectal, prostate, lung, breast cancers and glioblastomas. ASC protein is absent or downregulated in 62.5 per cent of the melanoma tissue samples and in 58.3 per cent of the investigated melanoma cell lines. Most melanoma cells are highly resistant to widely used chemotherapeutic drugs, thus novel pathways and targets need to be investigated for the development of effective therapies. We aimed to clarify the role of ASC in increasing the chemo-sensitivity of melanoma cell lines, which lack its expression. ASC was reintroduced into two of these cell lines, MeWo and Skmel 19, using a lentiviral infection system, which establishes the stable expression of ASC. Control GFP vector- and ASC vector- infected cells were treated with DNA damaging drugs - doxorubicin, etoposide and dacarbazine and the death receptor ligand TRAIL. In short term viability assays we have found that ASC expression confers slight chemosensitization to doxorubicin (Skmel 19) and etoposide (Skmel 19). A more pronounced effect was observed with TRAIL in Mewo cells. More importantly, we found in long term assays that re-expression of ASC in Mewo and Skmel 19 dramatically decreased their colony forming capacity.
In vivo studies of ASC specks as antigen delivery vehicles
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2016., 2016.) Yaşa, Seda.; Özören, Nesrin.
ASC is a 22 kDa adapter protein which is an essential component of the NLRP3, NLRC4 and AIM2 in ammasome complexes. ASC has critical functions in in ammatory and pyroptotic signaling pathways via activating caspase-1. In unstimulated cells, the ASC protein is soluble in the cytosol however upon stimulation it forms globular speck structures in close proximity to the nucleus. The importance of the ASC protein in the development of humoral immunity has been demonstrated upon vaccination of wild type and ASC knock-out mice with MF59-adjuvanted in uenza vaccine. Moreover, the importance of ASC specks in cell-to-cell communication by activation of other macrophages via phagocytosis of extracellular ASC specks after pyroptosis has been demonstrated. We previously reported that ASC specks can be loaded with the model antigen ovalbumin and OVA-loaded ASC specks can be puri- ed and fed to macrophages, which engulf the ASC-specks via phagocytosis and start degrading the protein constituents. Based on these informations, we have shown the long lasting stability of ASC specks inside the mice's bodies and its localisation to the important immune organ, spleen. Then, we checked the stimulation of immunity in in vivo mice studies using H5 (protein coat of H.In uenza virus) & OVA loaded ASC specks. Comparing the antigen speci c IgG titers, we demonstrated that we can use ASC specks as H5 antigen delivery vehicle against H5N1 virus. Also, our immunisation and tumor development experiments proved that ASC specks can be used as a novel adjuvant modality in vaccine technology. Anti-tumor e ect of ASC specks on tumor development provides an opportunity for us to improve ASC specks against tumor.
Investigation of homotypic PYD and CARD domain interactions in ASC speck assembly
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2019., 2019.) Otaş, Hasan Ozan.; Özören, Nesrin.
Apoptosis-associated speck-like protein containing a CARD (ASC) is a 22 kDa protein containing conserved PYD and CARD domains that belong to death-fold superfamily. Homotypic interactions between the domains occur via providing at least three certain surfaces of contact (Type I, II and III). ASC has an adaptor role between receptor and e ector proteins in the in ammation process having the ability to form a supramolecular globular complex called ASC speck through PYD and CARD homotypic interactions. When expressed in truncated form as PYD and CARD separately, these domains form lamentous structures. Apparently, these bers compact onto each other during the wild type ASC polymerization. In this study, our aim was to elucidate the importance of speci c locations on PYD-PYD and CARD-CARD interaction surfaces during the polymerization process. E ects of 19 single and 22 double mutations were observed introducing them on important residues such as E13A, D48A, Y60A, E130A, Y146A, R150A and M159A to hit one and/or two interaction surfaces at the same time. E ects of the mutations were visualized using uorescence and confocal microscopy to qualify the change in the level of organization of the phenotype from lamentous to soluble. Next, we analyzed rate of homooligomerizations in mutant sets using FRET technique to quantify the interaction e ciencies in presence of disruptive mutations. Our results showed that PYD mutants, which've been known to disrupt homooligomerization, are able to provide multimeric lamentous structures when expressed together with their wild type counterparts although CARD mutants have less tolerance to mutations in the interaction surfaces. We identi ed certain mutations that increase, decrease or block FRET signal. Our study provides better explanation and new insights about the oligomerization dynamics of the in ammasome complex.
Regulation of human BFK
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2014., 2014.) Uğurlu, Serkan.; Özören, Nesrin.
Bcl-2 protein family members are critical regulators for apoptosis. Reduced levels of apoptotic Bcl-2 family members are detected in gastrointestinal cancers which are responsible for a considerable part of the deaths from cancer. BFK (Bcl-2 Family Kin) is a novel pro-apoptotic Bcl-2 family member specifically expressed in the human gastrointestinal tract. BFK has the characteristic BH3 domain, which was shown to be essential for the apoptosis inducing activity of pro-apoptotic Bcl-2 family members. In the colon four alternatively spliced isoforms were identified. Human and mouse BFK genes share 70% homology at the DNA level and 68.7% homology at the aminoacid levels. Interestingly, human and mouse BFK genes show distinct expression patterns. To explain these differences, we performed gene evolution analyses on the promoter region as well as coding region of these genes. We found that the human BFK promoter experienced positive selective pressure and acquired a distinct set of repetetive elements and transcription factor binding sites. In this study, we identified several novel transcription factor candidates which may have roles in the transcriptional regulation of human BFK. As transcription factors, PARbZIP family members (especially TEF and NFIL3) regulate BFK upon binding to its promoter region. NFIL3 supresses TEF induced BFK transcription in HCT116 cells. We also studied hormonal regulation of human BFK. Tamoxifen, as a mixed agonist/antagonist of estrogen, upregulates human BFK levels in SW707 cells. We hope this study contributes to a better understanding of Bcl-2 family.
Study of the polymerization dynamics of ASC and its domains
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2017., 2017.) Kuzucu, Hulusi Onur.; Özören, Nesrin.
Apoptosis-associated speck-like protein containing a CARD (ASC) is a 22 kDa protein composed of two conserved death-fold domains, PYD and CARD, that act as an adaptor protein in in ammasomes, multimolecular complexes that promote the maturation of pro-in ammatory cytokines IL-1 and IL-18. Death-fold domains are known to mediate the formation of higher order complexes through three distinct type of interactions. The importance of these interaction types are distinguished through ASC's ability to form a high molecular globular complex called the ASC speck. It has been shown that PYD-PYD and CARD-CARD homotypic interactions between ASC molecules is necessary to form this structure. Our group has reported the signi cance of homotypic PYD interactions, as well as of the residues on interaction surfaces. In this study, we have revisited the dynamics of homotypic PYD interactions by observing the formation of lamentous structures between mutant and wildtype variants. It was observed that PYD mutant variants previously known to disrupt homooligomerization such as E13A, K21A, K26A and D51A, were still able to contribute to multimeric structures along with their wildtype counterparts. Then, we have focused on the importance of homotypic CARD interactions, and investigated the signi cance of certain residues in CARD homooligomerization through Forster Resonance Energy Transfer (FRET) analysis. We have observed that the stability of interaction surfaces is more important in CARD, and mutant variants D134A, M159A and R160A showed a complete lack of lament formation. Lastly, we have selected wildtype CARD and CARD E130A mutant variant and done Single Molecule Force Spectroscopy (SMFS) to measure the physical strength of homotypic CARD interactions, and how disruption of interaction surfaces a ect said strength.
Two faces of ASC : central innate immunity adaptor ASC turns into melanoma ally and novel mechanism demistifying dsRNA induced NLRP3 inflammasome activation through Bax/Bak mediated cell death
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2011., 2011.) Özkurede, Varol Ulaş.; Özören, Nesrin.
In this study, by specifically focusing on the role of ASC in melanoma and NLRP3 activation in response to double stranded RNA, we try to provide answers to more general but basic questions: “How and why would cancer cells alter the function of inflammatory proteins?” and “How can NLRP3 inflammasome recognize varying PAMPs and DAMPs with totally different natures and molecular structures?”, respectively. Although having mRNA expression for all necessary NLRP3 inflammasome components, healthy primary melanocytes showed no response to NLRP3 activating signals as measured by IL-1 beta secretion via ELISA. Whereas ASC was silenced at the transcription level, the protein was present and functional in certain melanoma cell lines. Moreover, knock-down of Asc in these cell lines by shRNA resulted in diminished NLRP3 activation. Overall, our observations support the idea that cancer cells may activate the nonfunctional inflammasome machinery, supposedly to increase cytokine secretion which may help angiogenesis and subsequently metastasis. In the second part of our study, we investigate dsRNA recognition mechanism and pathway leading to the NLRP3 inflammasome activation. Treating LPS primed Ips-1/Trif DKO macrophages we observed that Ips-1 is required for intracellular pIC recognition. Inflammasome activity analysis of Bax/Bak DKO macrophages and immunofluorescence assay for active Bax showed that, Ips-1 acts on apoptotic Bcl2 family members Bax/Bak triggering programmed cell death mechanism. Resulting membrane permeabilization causes loss of cytosolic K+ ion which is sensed by NLRP3 leading to NLRP3 inflammasome activation. Our data provides the lacking unifying model of NLRP3 activation mechanism: Any pathogenic organism or danger signal causes loss of membrane integrity. Resulting damage disturbs the active regulation of cytosolic ion balance, causing loss of K+ from cytosol, which is sensed by NLRP3 inflammasome resulting in NLRP3 inflammasome activation.

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Browsing Moleküler Biyoloji ve Genetik by Subject "Apoptosis."

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