Theses supervised by Prof. Dr. İbrahim Halil Kavaklı
17 theses · Koç University
Characterization of the single nucleotide polymorphisms that effect repression activity and stability of cryptochrome2
The daily rhythm of sleep/wake cycles, body temperature, metabolism, hormone secretion and many other physiological activities are in control of the circadian mechanism which is conserved from simple cyanobacteria to complex humans. In mammals, the circadian clock is endogenous and highly regulated with complex transcriptional and translational feedback loops (TTFL). Entrainment of the rhythm through the whole body is maintained by suprachiasmatic nuclei (SCN) located in the anterior hypothalamus with the cues from the environment. Many physiological processes are linked to the circadian clock, and it is not surprising that disruption of the clock can lead to certain disorders as demonstrated in various studies. At the molecular circadian mechanism of mammals, Circadian Locomotor Output Cycles Kaput (CLOCK) and Brain and Muscle ARNT-Like-1 (BMAL1) heterodimerizes and transactivates the expression of clock controlled genes including Period (Per) and Cryptochrome (Cry) by binding to E-box(CACGTG). PER and CRY accumulate in the cytoplasm and translocate into the nucleus, where they repress BMAL1/CLOCK-driven transactivation. The inhibition of BMAL1/CLOCK is relieved by the breakdown of CRY and PER proteins, resetting the cycle. In this study, the effect of the rare CRY2 variants in the central clock mechanism is aimed to be investigated. To this end, the fifteen rare Cry2 variants are identified from the database of 1000 Genomes Project and Ensembl. The initial assessment of these fifteen variants is predicated to be pathogenic. Further experimental and structural studies lead us to study p.Pro123Leu CRY2, p.Ser210Ile CRY2, and p.Asp406His CRY2 variants, which are located at a functionally important domain of the CRY2 called the secondary pocket, plays a role in CLOCK binding. CRY2 variants were unable to repress BMAL1/CLOCK driven transcription. Further biochemical studies indicated these variants were less stable than the wild type CRY2. Additionally, compared to wild type CRY2 the p.Pro123Leu CRY2, the p.Asp406His CRY2 and p.Ser410Ile had reduced affinity to CLOCK assessed by co-immunoprecipitation. Finally, it has been observed that p.Ser210Ile CRY2 and p.Asp406His CRY2 are not capable of rescuing the circadian rhythm in Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cell line by complementation test. Collectively our data suggest that the secondary pocket of CRY2 plays a significant role not only for the CLOCK binding but also for the stability and the proper circadian rhythm at the cellular level.
Stabilization of the CLOCK:BMAL1 heterodimer decreases circadian rhythm amplitude
Rhythmic changes in behaviour and/or physiology are governed by the daily light-dark cycle in most organisms to increase their fitness to the environment. These rhythmic changes are generated from the biological clock and last about 24 hours. In mammals, a hierarchically ordered circadian clock that is highly regulated by complex transcriptional-translational feedback loops (TTFLs) is observed. At the molecular level, CLOCK and BMAL1 form a heterodimer to bind E-box sequences within the promoter region of the clock-controlled genes (including Cryptochrome (Cry) and Period (Per)) to initiate the transcription. Then CRYs and PERs which are accumulated within the cytosol, translocate into the nucleus along with Casein Kinase Iε to represses BMAL1:CLOCK-driven transcription. Small molecules regulating the activities of these core-clock proteins could offer temporal control over the circadian clock. Considering the pathologies- such as cancer, metabolic diseases, and accelerated aging- that can arise from the lack of a robust circadian clock, identification of such molecules carries great importance. Therefore, I characterized the CLK95, which is previously identified by our group, as a novel CLOCK and BMAL1 binding small molecule. I showed that CLK95 stabilizes the interaction between CLOCK and BMAL1 which, in turn, enhances their nuclear localization both in vitro and in vivo. Further experiments revealed that the CLK95 dampens the amplitude and increases the period of the circadian rhythm by stabilizing the positive loop of the primary TTFL. Considering elevated CLOCK and BMAL1 levels inhibit cell growth, CLK95 may show therapeutic effects against cancer cells due to its enhancing effect on CLOCK and BMAL1.
Memeli sirkadyen saatinde kriptokrom 1'i stabilize eden bir küçük molekülün karakterizasyonu
In most organisms, several physiological and behavioral mechanisms are regulated in a circadian rhythm-dependent manner. The mammalian circadian clock is generated by negative and positive transcriptional-translational feedback loops (TTFL), which consists of BMAL1, CLOCK, Periods (PER) and Cryptochromes (CRY) at the molecular level. At the positive arm of the TTFL loops, BMAL1 and CLOCK form a dimer and bind to E-box elements to initiate the transcription of the clock-controlled genes (CCGs) including the Per and Cry genes. Within the time Cry1/2 and Per1/2/3 accumulate in the cytosol and then translocate into the nucleus along with Casein kinase I epsilon/delta. They interact with BMAL1:CLOCK heterodimer and repress their activity and, in turn, repress the transcription of the CCGs. In mammals, 43% of the protein-coding genes are clock-controlled and transcribed rhythmically in at least one tissue. Therefore, disruption of the circadian clock as a result of circadian misalignment or genetic factors will be expected to greatly affect most of the physiological systems. In fact, several different diseases are shown to be linked to circadian disruption such as sleep disorders, metabolic and neural diseases, and cancer progression. That's why several groups have initiated drug discovery studies to find molecules that regulate the circadian clock for the treatment of these diseases. In this thesis, I characterized a novel small molecule (M54) specific for the core clock protein CRY1 in vitro level. M54 is discovered by an in silico approach called structure-based drug designing (SBDD) and predicted to bind to the primary pocket of CRY1. The primary pocket is important for the regulation of CRY stability due to interactions with the E3 ubiquitin ligases (FBXL3 and 21). I showed that M54 significantly increases the circadian period in a dose-dependent manner at the cellular level. Further biochemical studies demonstrated that M54 increases the stability of the CRY1. This study offers a new avenue as a therapeutic approach for patients who suffer from circadian clock-related disorders associated with dampened CRY1 levels.
1000 genom projesinde yer alan CLOCK geni tek nükleotid polimorfizmlerinin in silico ve in vitro analizleri
Circadian rhythm is an internal process regulating ~24-h physiological and behavioral processes in organisms. In mammals, circadian rhythm is generated by transcription and translational feedback loop (TTFL) mechanism as a result of the interaction between core clock proteins. In TTFL, CLOCK and BMAL1 interact with each other and form a heterodimer to bind E-box sequences within the promoter region to initiate the transcription of the clock-controlled genes, including Period (Per) and Cryptochrome (Cry). Within the time, CRYs and PERs accumulate in the cytosol and then translocate into the nucleus with Casein Kinase Iε and repress BMAL1: CLOCK driven transcription. There are other auxiliary TTFLs exist that control circadian rhythm. Genetics and epidemiolocal studies suggest factors that disturb circadian rhythm result in susceptibility or may directly cause several diseases such as obesity, diabetes, cardiovascular diseases, aging, cancer, mood, and sleep disorders. Several single nucleotide polymorphisms (SNPs) for core clock genes have been identified and shown to be associated with different types of diseases. However, whether these SNPs contribute to the different type of the disease are ill-defined. One of the challenges in these approaches is that genome-wide association sequences (GWAS) studies have limitations in functional prediction. To address that, I developed using in vitro studies following the in-silico techniques to identify and characterize functional CLOCK SNPs from 1000 Genomes Ensemble to show the effect of a particular missense mutation on CLOCK protein on function. Such systematic approaches would allow us to discover SNPs with pathological effects and understand how these SNPs affect proteins' function. In this thesis, I performed a functional characterization of rare CLOCK missense variations (p.Phe104Cys, p.Leu118Arg, p.Asp119Val, p.Gly120Val, and p.Phe121Cys) identified from the Ensembl database. I initially analyzed these variations using computational tools. Results revealed that variants are located on the functionally important region of CLOCK. I used in vitro experimental approach and showed p.Leu118Arg, p.Asp119Val, and p.Phe121Cys CLOCK had reduced transactivation activity while p.Gly120Val CLOCK had increased transactivation along with BMAL1. However, p.Phe104Cys CLOCK had not been comparable transcriptional activity. To attrubitue these functional difference on the affinity between CLOCK SNPs and BMAL1, I performed co-immunoprecipitation between them Results indicated that p.Leu118Arg, p. Asp119Val, p.Gly120Val CLOCKs had reduced affinity to BMAL1and interestingly p.Phe121Cys CLOCK had increased the affinity to BMAL1. To gain more insight I further showed that the CRY1 had reduced repressor activity on p.Leu118Arg and p.Phe121Cys CLOCKs. Meanwhile, the estimation binding energy analysis of MD simulations supported the biochemical results, and binding energy analysis per residues was used to examine the mechanism of such SNPs effects. Collectively, I discovered that even single nucleotide changes in CLOCK directly affect the CLOCK functions. Hence, illumination of the effects of CLOCK SNPs would also help develop novel treatment strategies for diseases related to clock disruption for further studies and provide valuable information for the structure-function of CLOCK in circadian clock mechanism.
Functional characterization of p.Ser420Phe CRY2 variant
The circadian clock is an innate mechanism that enables organisms to adapt to the rhythmic changes of the environment. In mammalian cells, this mechanism is achieved by the interaction of four core clock proteins (CRYs, PERs, CLOCK, and BMAL1). Human clock-gene variations are associated with numerous behavioral and physiological diseases, such as sleep, metabolism, addiction, neurological disorders, and endocrine and metabolic. However, little is known about the possible effects of variations at the molecular level. Currently, the functional consequence of these variations and the strength of their association with the disease remains unclear. The functional analyses of variants help us to understand the role of amino acid residues in the structure and function of proteins. In this study, rare CRY2 variants were selected from the 1000 Genomes Project, and the Ensembl database. SNPs were filtered based on the prediction of pathogenicity using in silico tools. Using the 3D structure of the CRY2 protein, the variant (p.Ser420Phe CRY2) located in the vicinity of the coiled-coil-like helix (CC helix) was functionally characterized. We have found that the p.Ser420Phe CRY2 had reduced repression activity on CLOCK/BMAL1-driven transcription and showed rescued circadian rhythm with a shorter period than wild type in Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cell line. Moreover, the variant had reduced affinity to other core clock proteins CLOCK and PER2 and could not properly localize in the nucleus using biochemical methods. Further stability results suggested that the p.Ser420Phe CRY2 variant was degraded by a noncanonical mechanism. Collectively, our results show that the CC helix region plays an important role in the degradation of CRY2, interaction with other core clock proteins, also nuclear shuttling.
Identification of ABE2A as a circadian amplitude enhancer with drug-like potency
Many species have an adaptation system to the period of the solar day. This endogenous mechanism is called the circadian clock, which can be defined as an autonomous 24h cycle controlling daily rhythms in physiology and behavior. Thus, a robust circadian clock is required for many processes in our body to work smoothly. Our body's control center of circadian clock operation is SCN (suprachiasmatic nucleus), which uses the phoetic input sensed by intrinsically photosensitive retinal ganglion cells (ipRGCs). SCN regulates cellular clocks and peripheral tissues and coordinates them with the help of neural or hormonal signals. This oscillatory system depends upon transcriptional translational regulatory feedback loops (TTFLs). The main loop consists of CLOCK, BMAL1 (positive arm), and CRYs, PERs (negative arm). Mechanistically, CLOCK and BMAL1 interact with each other in the cytosol. Then this heterodimer translocates into the nucleus and binds to E-Box elements within the promoter of CRYPTOCHROME (CRY) and PERIOD (PER). Cryptochrome (CRY), PER, and the epsilon isoform of casein kinase I (CK1ε) form a complex in the cytosol that enters the nucleus and inhibits their own transcription regulated by CLOCK and BMAL1 heterodimer. Any disruption in this mechanism may result in diseases or disorders. Small molecule modifiers are efficient tools to repair this perturbed mechanism and have many advantages over other therapeutic approaches. In my study, I characterized a novel small molecule called ABE2A, 100 times more effective derivative of the CLK8 molecule, which increases the amplitude of circadian rhythm by specifically interacting with CLOCK and decreasing the interaction between CLOCK and BMAL1. Further experiments revealed that ABE2A decreases the nuclear abundance of CLOCK, BMAL1 and increases the cytosolic abundance of BMAL1. Additionally, it reduces the total cell protein level of CLOCK and increases PER2. ABE2A also affects the transcriptional levels of E-box and D-box-regulated genes. For instance, it drops the expression of core clock genes such as DBP, BMAL1, and PER2. Overall, similar to CLK8, ABE2A reduces the positive arm components in the nucleus, limiting the transcription of the negative arm. Thus, the repression activity in the TTFL is stabilized, and the amplitude of the circadian rhythm enhances. For these reasons, and also by being effective at nanomolar doses, ABE2A has a drug-like potency against various health problems related to decreased amplitude, such as some metabolic diseases, mood disorders, and accelerated aging.
Discovery of small molecule: Regulates the cryptochrome stability and controls blood glucose levels in diabetic mice
The circadian rhythm controls the behavioral, biochemical, and physiological variables in most living organisms, from bacteria to mammals. The circadian rhythm is an endogenous, the most well-known biological rhythm, and refers to a 24-hour cycle that regulates various physiological processes like hormone secretion, food intake, sleep-wake cycles, body temperature regulation, and metabolism. The mammalian circadian clock mechanism is endogenous, although it is synchronized with the environmental cues through the suprachiasmatic nuclei (SCN) located in the anterior part of the hypothalamus. Circadian clock disruption influences metabolic health and is closely associated with many diseases, including sleep disorders, neurological conditions, immune dysregulation, obesity, diabetes, and cancer. Cryptochromes (CRYs), one of the core clock proteins, are transcriptional repressors of the circadian clock in mammals. The stability of CRYs is important because of the ability to alter the period and amplitude of the circadian rhythm. The gluconeogenesis pathway is regulated by a variety of factors, including hormones such as glucagon and insulin and the circadian clock mechanisms. On the molecular level, CRY interacts directly with the glucagon-mediated G-protein coupled receptors, in turn, inhibits the gluconeogenesis pathway that controls glucose metabolism. Therefore, we aimed to discover small organic molecules which increase the stability of CRYs and inhibit gluconeogenesis. Such molecules could be used as anti-diabetic drugs, which control blood glucose levels. To this end, we used a structure-based drug design approach against the primary pocket of CRY, which is responsible for their degradation. Around 2 million small molecules with non-characterized functions were screened through molecular docking in silico. The candidate small molecules were tested for cellular toxicity and their effects on CRYs stability, circadian clock, and gluconeogenesis. We identified a novel molecule, TW68, which increases the stability of both CRY1 and CRY2, lengthens the period of circadian rhythm, and represses gluconeogenic genes in further characterization of pre-clinical studies. In transgenic and fat-induced diabetic animal models, TW68 regulated the fasting blood glucose levels in biochemical and physiological studies. These results signify the therapeutic potential of TW68 on the circadian clock – gluconeogenesis-related metabolic diseases, including type 2 diabetes mellitus.
In vitro characterization of CLOCK-interacting small molecules that changes the phase of the circadian rhythm
Circadian rhythms, the intrinsic 24-hour biological cycles, govern a myriad of physiological processes, including sleep/wake sequences, hormonal oscillations, thermoregulation, and blood pressure fluctuations. These rhythms are predominantly conserved across a broad spectrum of organisms, from prokaryotic cyanobacteria to complex eukaryotes such as mammals and plants, and they enhance an organism's adaptive capabilities in relation to their environmental context. Within mammals, the regulation of these rhythmic patterns occurs on two primary levels: the organismal level, where the master clock situated in the suprachiasmatic nuclei (SCN) synchronizes the circadian clock through a delicate interplay of neuronal and hormonal cues, and the molecular level, where intricate transcriptional-translational feedback loops (TTFLs) modulate the clock mechanism. Key proteins such as CLOCK, BMAL1, CRYs, and PERs are integral to the functionality of the clock mechanism. The protein interaction begins with transcription factors CLOCK and BMAL1 forming a heterodimer that binds to the E-BOX of clock-controlled genes, such as Cry and Per, thereby initiating their transcription. Following accumulation of PERs and CRYs in the cytosol, a heterodimer forms and, upon binding with Casein Kinase Iε, translocates into the nucleus to inhibit CLOCK:BMAL1 driven transcription. Subsequent degradation of CRYs and PERs through ubiquitin-dependent proteasomal degradation lifts this transcriptional repression, triggering the commencement of a new cycle. Disruptions to this carefully orchestrated rhythm can have significant health implications, potentially precipitating severe pathologies like metabolic and cardiovascular diseases, cancer, sleep disorders, depression, and Alzheimer's disease. Consequently, the search for small molecules capable of regulating the circadian clock holds promising potential. The objective of this study is to identify novel small molecules that can engage, and subsequently modulate the activity of the CLOCK protein, utilizing structure-based drug design. Following an in silico analysis of millions of molecules, candidate drug molecules were chosen through a comprehensive evaluation process. These selected molecules were synthesized and subjected to in vitro screening to assess their circadian rhythm attributes. The resulting phase-altering small molecules were then further characterized to elucidate their potential in treating circadian rhythm-related pathologies.
Allosteric regulation and functional dynamics of CRY1 in the mammalian circadian clock: Insights from mutational and small molecule interactions
Cryptochromes (CRYs) are essential components of the molecular clock that governs circadian rhythms in mammals, functioning as inhibitors of BMAL1/CLOCK-driven transcription. While mammals possess two CRYs with distinct roles in the circadian clock, the mechanisms underlying their differential functions remain unclear. This study elucidates how a specific mutation in CRY1 allosterically alters its dynamic behavior. Molecular dynamics simulations were conducted on eight CRY1 mutants experimentally shown to exhibit reduced repressor activity. My findings reveal that mutations in CRY1 affect the dynamic behavior of the serine loop and the accessibility of the secondary pocket, changes not observed in CRY2. Further analysis highlights that the flexibility of the serine loop influences secondary pocket volume, with S44 and S45 playing crucial roles through their interactions with E382. Additionally, I explored the potential for small molecules to modulate CRY1 allosterically, demonstrating both in silico and in vitro that this regulation impacts CRY1's affinity to CLOCK. This discovery provides a promising avenue for developing therapeutics targeting CRY1, though it also raises concerns about unforeseen side effects of existing small molecules. To address this, virtual screening was performed to identify compounds targeting the CRY1 secondary pocket. Subsequent phenotypic analysis revealed five distinct effects on circadian rhythm, underscoring the complexity of this regulatory mechanism. This study offers critical insights into CRY1 dynamics, its interaction with CLOCK, and the potential for small-molecule modulation, paving the way for advancements in circadian biology and drug design.
Memeli CRY 'lerinin ikincil ceplerini hedefleyen küçük bir molekülün in vitro karakterizasyonu
Sirkadiyen ritimler, çeşitli fizyolojik ve davranışsal süreçleri düzenleyen, yaklaşık 24 saatlik endojen döngülerdir. Bakterilerden memelilere kadar çeşitli organizmalarda bulunurlar. Sirkadiyen ritimlerin oluşumu ve sürdürülmesi, sirkadiyen saat olarak adlandırılan dahili bir zaman tutma sistemi tarafından sağlanır. Memelilerde, hipotalamusta bulunan suprakiasmatik çekirdek (SCN) ana saattir. SCN, retinadan çevresel ışık ipuçlarını alır ve sirkadiyen zamanlamayı düzenler. Neredeyse her doku ve hücrede bulunan çevresel saatler, SCN tarafından sırayla senkronize edilir. Çevresel saatler otonom olarak salınabilse de, çevreyle uyum sağlamak için beslenme zamanları ve sıcaklık gibi dış ipuçlarına güvenirler. Moleküler düzeyde, sirkadiyen saat, dört ana çekirdek saat proteinini içeren transkripsiyon-translasyon geri bildirim döngüleri (TTFL'ler) tarafından yönlendirilir: CLOCK, BMAL1, PER'ler ve CRY'ler. CLOCK:BMAL1 kompleksi, baskılayıcıları olan PER'ler ve CRY'lerin transkripsiyonunu başlatır ve bu da CLOCK:BMAL1 aracılı Ekutusu transkripsiyonunu ve dolayısıyla transkripsiyonlarını baskılar. Sirkadiyen ritimlerin genetik mutasyonlar, düzensiz ışık maruziyeti veya yaşam tarzı nedeniyle bozulması, metabolik, kardiyovasküler, nörodejeneratif, uyku bozuklukları ve kanser dahil olmak üzere çeşitli bozukluklarla ilişkilidir. Son çalışmalar, saat proteinleri ve bunların translasyon sonrası modifikasyonları da dahil olmak üzere sirkadiyen saatin farklı yönlerini hedefleyen küçük molekül modülatörleri keşfetmiştir. Bu moleküller, sirkadiyen zamanlamayı düzeltmek ve sağlık sonuçlarını iyileştirmek için terapötik bir potansiyel sunmaktadır. Bu çalışmada, CRY1'in ikincil cebini hedef alan A7 olarak adlandırılan yeni bir küçük molekül tanımladık ve karakterize ettik. A7, genliği arttırır ve ikincil cebi hedef alarak CLOCK:BMAL1 aracılı transkripsiyonda CRY1'in baskılama aktivitesini azaltır. Ayrıca, DBP, PER2, CRY1 ve BMAL1'in transkripsiyonel seviyeleri A7 uygulaması ile artar. A7'nin çekirdek saat proteinlerinin hücre içi lokalizasyonu üzerinde herhangi bir etkisi yoktur. Daha da önemlisi, CRY1 ve CLOCK arasındaki etkileşimi bozar. Sonuçlarımız, A7'nin CRY1 ve CLOCK'a bağlanmasına müdahale ederek sirkadiyen saati düzenlediğini göstermektedir. Bu bulgular, azalmış genlikle ilişkili sağlık sorunları için yeni bir tedavi yaklaşımı sunmakta ve çekirdek saat protein etkileşimlerinin ve bunların sirkadiyen ritimler üzerindeki etkilerinin daha derinlemesine anlaşılmasını kolaylaştırmaktadır.
Mavi ışığın tek hücreli organizmaların transkriptom profiline etkisi
The ability of light perception is crucial for the survival of most organisms that enables them to adjust their physiology and metabolism to the changing environmental conditions. Light, in contrast, is also a threat to any living organism owing to the deleterious effects it can have on nucleic acids, lipids and proteins. Therefore, the capacity to sense and respond to light is widespread among prokaryotes and eukaryotes to survive and adapt themselves as a result of selective pressure of the solar irradiation. This dissertation concentrates on the analysis of light regulated pathways in unicellular organisms, non-phototrophic prokaryote Vibrio cholerae and phototrophic red alga Cyanidioschyzon merolae, via transcriptome profiling. C. merolae is one of the most primitive of photosynthetic eukaryotes. Since it is an extremophile, it is conceivable to study the effect of light on this organism to see how it adapts itself to different environmental conditions and to establish an evolutionary conserved global light response between algae and land plants. Therefore, we decided to investigate the direct effect of red and blue lights at the transcriptional level as well as to verify known blue light receptor genes and the involved transduction pathways using next generation RNA-seq approach. Our results indicated that transcriptional regulations of 35 % of the total genes, including the genes encodes 46 % of transcription factors, were regulated by blue and red lights in C. merolae. Unexpectedly, although there are yet no identified red light photoreceptors, 22 % of the total genes (1116) were regulated by the red light; 521 genes were solely red-light responsive. Transcriptional modulation due to light exposure does not arise from photo-oxidative stress. Blue light dependent regulation of three cryptochromes (CmPHR2, CmPHR3 and CmPHR7) implies a potential role in light-dependent transcriptional regulation in C. merolae. In spite of absences of any red light photoreceptors, a great impact of the red light on the biological processes may suggest the importance of retrograde signaling in this organism. Secondly, we investigated the effect of blue light in non-phototrophic bacteria V. cholerae by using genetics and transcriptome profiling. Genome-wide analysis revealed that the transcription of 6.3% of the genes was regulated by blue light in V. cholerae. To understand signaling mechanisms, we generated several knockout cell lines and subjected to genome-wide analysis under blue light condition. Studies with a double mutant confirm an anti-sigma factor (ChrR) and a novel putative metalloregulatory-like protein (MerR) are responsible for the genome-wide regulation to blue light response in V. cholerae. We further showed that blue light enhances ROS production, possibly generated through the oxidative phosphorylation pathway. These results demonstrate that V. cholerae responds to blue light with a novel mechanism to produce an appropriate response against photo-oxidative stress. This response regulates the transcription of genes involved in cellular protection, DNA repair, and carbon metabolism. Outside its host, V. cholerae can survive for extended periods in natural aquatic environments. Therefore, the regulation of light response for V. cholerae is a critical cellular process for its survival. Collectively, all these data reveal that light is important not only for phototrophs but also non-phototrophs to regulate their metabolism and related physiological pathways through elegant and complex signaling cascades.
CLOCK-BMAL1 transaktivasyonunu etkileyen yeni transkripsiyon faktorlerinin tanımlanması
Circadian clocks are self-sustained time-keeping systems that generate circadian rhythms with a period of approximately 24 hours. Circadian clocks are internal pacemakers that influence human physiology, endocrinology, xenobiotic detoxification, cell growth, and behavior. In mammals, the circadian clock mechanism involves several proteins that participate in positive and negative transcriptional feedback loops. Proteins involved in the positive feedback loop include BMAL1 and CLOCK proteins. These proteins form heterodimers and bind to E-box elements (CACGTG) in promoter of period (Per), cryptochrome (Cry) and other clock-controlled genes. PER and CRY proteins form heterodimers that interact with casein kinase I ε (CKIε) and then translocate into the nucleus where CRY acts as a negative regulator of BMAL1:CLOCK driven transcription. Genetic studies on mouse indicated that indeed there are more core clock components to regulate core clock at the molecular level. To identify components that have an effect on the BMAL1:CLOCK transactivation, high-throughput luciferase reporter assay was utilized to screen 1400 mammalian transcription factors. Initial screening showed that WW domain-containing transcription regulator protein 1 (WWTR1) is one of the top candidates that showed high repression activity for BMAL1:CLOCK driven transcription on Per1 promoter. Herein, I demonstrate that this repression activity is achieved by physical interaction of the WWTR1 with Bmal1 protein with co-immunoprecipitation and bi-molecular fluorescence complementation assay (BiFC). To see its effect on the circadian clock, Wwtr1 was downregulated by shRNA in NIH3T3 Per1: dluc and U2-OS Bmal1: dluc cell lines. There was damping in amplitude and advance in phase of oscillation of rhythm in both cell lines. Additionally, knockdown of Wwtr1 affected the transcriptional regulation of the core clock genes, especially transcription of the Bmal1 and Cry1 genes. Furthermore, WWTR1 appears to acts as co-activator on Cry1 and Bmal1 promoters. ChIP analysis also demonstrates WWTR1 occupation on Cry1 promoter. Collectively all these results suggest a potential new core clock component, WWTR1, for the regulation of circadian rhythms by repressing BMAL1:CLOCK transactivation and by regulating Bmal1 and Cry1 transcriptional level.
CRY1 ve CBS Etkilesimin Sirkadyen Ritim ve Metabolizma Uzerindeki Iki Yonlu Etkisi
Circadian clock controls the physiology and behavior in order to synchronize them to 24-hr rhythm. Synchronization of physiological events in our body both prevents us from diseases and helps us to overcome the existing problems. Unlike the central clock located in the SCN (suprachiasmatic nucleus) which is regulated by light, peripheral clocks either regulated by central clock or by feeding cues. Despite the many efforts have been done in the field to understand how transcription and translation is controlled by circadian clock, they are not enough to explain the oscillation of 50% of the metabolites. There is little known about how circadian clock molecular machinery controls metabolism or post-translational regulations of core clock components to regulate metabolically-important proteins. Here we characterize a novel molecular interaction between one of the negative arm proteins of circadian clock (CRY1) and an important metabolic enzyme CBS (cystathionine-beta-synthase). Mutations resulting loss-of-function of CBS causes a multi-systemic disorder called homocystinuria. Physical interaction of these two proteins provides bi-directional regulation of both circadian rhythm and metabolism. Other than transcriptional repressor role of CRY1, as a novel post-translational regulatory mechanism, it binds to CBS and regulates metabolism by favoring oligomerization and increasing the activity of it. On the other hand, wild type CBS, but not disease-causing mutant CBS, binds to CRY1 and enhances the repressor activity. Additionally, we also analyzed the effect of mutant CBS which causes homocystinuria in humans. CBS-I278T cannot form oligomers and thus, doesn't have any enzyme activity. This single mutation blocks the interaction of CRY1 and CBS. We verified the physiologic importance of this interaction by performing untargeted metabolomics screen in Cry1-/- and Cbs-/- liver samples and analyzed the significantly affected pathways. Strikingly, Cry1-/- affects the same pathways that are affected by Cbs-/-. These pathways are transsulfuration pathway, lysine, arginine and proline metabolisms and urea cycle. Our results provide better understanding about how circadian clock and metabolism regulate each other at post-translational level, with direct protein-protein interactions. We anticipate that our results will provide new circadian perspective to treat CBS-caused homocystinuria disease.
The effect of small molecule, CLK8, on circadian clock mechanism
Circadian rhythms are metabolic, behavioral and physical changes which repeat themselves with about 24-hour period. These rhythms arise from transcriptional-translational regulatory feedback (TTFL) loops at cellular level. In the positive arm of the TTFL, CLOCK and BMAL1 heterodimerize and activate the expression of the Per and Cry genes. In the negative arm of the loop, PER2:CRY1 complex repressed their own transcription by inhibiting transactivation of CLOCK:BMAL1 complex. Properly functional circadian clock is crucial for health as its perturbation leads to severe pathologies including metabolic and cardiovascular diseases, sleep and mood disorders, accelerated aging and cancer progresses. Therefore, discovery of small molecule modifiers of circadian clock is important for the development of potential interventions to repair circadian clock, in turn, to improve health. In this study, structure-based chemical genetic approach was used to find small molecules that specifically bind to CLOCK. Following the assessment of candidate small molecules by virtual screening according to changes in circadian rhythm, biochemical assays pointed out a compound (CLK8) as a circadian amplitude enhancer which specifically binds to CLOCK protein and modulates the interaction between CLOCK and BMAL1. Both in vitro and in vivo studies revealed that CLK8 reduces the nuclear abundance of CLOCK, overall expression of core clock genes and the protein levels of CLOCK and BMAL1 without altering the protein amount of PER2 and CRY1. These outcomes suggest that CLK8 enhances the amplitude of circadian rhythm by altering the stoichiometry among the core clock components in a way that the repression activity of negative arm of TTFL is stabilized by reducing the positive arm of the TTFL in nucleus. This study identifies CLK8 as a tool to understand the function of CLOCK in the regulation of circadian amplitude and as a potential candidate for therapeutic development for health problems associated with reduced circadian amplitude such as mood disorder, chronic metabolic diseases and accelerated aging.
Memeli sirkadiyen saatinde kriptokromu stabilize eden küçük moleküllerin keşfi ve karakterizasyonu
Sirkadiyen saat mekanizması neredeyse bütün canlılarda bulunur ve uyku-uyanıklık döngüsü, metabolizma, vücut sıcaklığı gibi birçok fizyolojik sürecin günlük ritminin korunmasında görev alır. Memelilerde sirkadiyen saat mekanizması organ ve dokularda içsel olarak bulunmakla beraber hipotalamusta bulunan suprakiazmatik çekirdek (SCN) tarafından senkronize edilir. Fizyolojik süreçlerin birçoğunun sirkadiyen saat ile bağlantılı olmasından dolayı, birçok çalışmanın da gösterdiği üzere, biyolojik saatin bozulması bazı hastalıklara yol açabilmektedir. Moleküler düzeydeki sirkadiyen saat mekanizması transkripsiyonel – translasyonel geribildirim döngülerinden oluşur. Merkezdeki döngüde BMAL1 ve CLOCK transkripsiyon faktörleri heterodimer oluşturarak Cry1/2 (Cryptochrome) ve Per1/2 (Period) genlerinin ekspresyonunu etkinleştirir. Daha sonra CRY ve PER proteinleri bir kompleks oluşturarak çekirdeğe geri dönerler ve burada BMAL1:CLOCK kompleksinin aktivitesini baskılayarak kendi ekspresyonlarını da baskılarlar. CRY1 ve CRY2 proteinlerinin degredasyonu ve stabilitesi SCF-FBXL3 ve SCF-FBXL32 ubikitin ligazları tarafından ayarlanır. CRY proteinlerinin stabilitesi önemlidir; çünkü sirkadiyen ritmin periyodunu ve genliğini değiştirebilir ve böylece metabolizmayı etkileyebilir. Bu tezin amacı CRY proteinlerinin stabilitesini arttıran küçük molekül ilaçların taranarak tip 2 diyabet tedavisinde kullanılmak üzere belirlenmesidir. Glikoz metabolizması diğer birçok mekanizma gibi sirkadiyen saat mekanizmasının kontrolü altındadır. Tip 2 diyabet veya diğer glikoz mekanizması bozuklarının tedavisinde kullanılmak üzere küçük molekül ilaçların keşfi için glukoneogenez yolağına odaklanılabilir. CRY1 protein seviyesi artışının etkisiyle glukagona bağlı glukoneogenezin inhibe edildiği önceki çalışmalarda görülmüştür. Bu inhibisyonun nedeni CRY1'in G proteininin α alt ünitesine bağlanarak GPCR'a bağlı cAMP artışının engellenmesidir. CRY proteininin stabilizörlerinin belirlenmesi için laboratuvarımızda daha önceden yaklaşık 2 milyon küçük molekül bilgisayar ortamında taranmıştır. Bu taramada moleküllerin CRY üzerindeki FBXL3/21 bağlanma bölgesi ile etkileşimine odaklanılmıştır. Bu tezin konusu olarak taramanın sonucunda elde edilen 32 tane küçük molekül ilaç adayının CRY stabilitesine, sirkadiyen ritme ve glukoneogeneze olan etkileri test edilmiştir. Bu adaylardan TW63 ve TW68 kodlu moleküller CRY1/2 proteinlerinin stabilitesini arttırmış ve U2OS Bmal1-dLuc hücrelerinde sirkadiyen ritmin periyodunu uzatmıştır. Buna ek olarak, glukagona bağlı glukoneogenez sırasında TW68 uygulanan HepG2 karaciğer hücrelerinde Pck1 ve G6pc glukoneogenez genleri baskılanmış ve glikoz üretiminde azalma görülmüştür. Bu sonuçlar, bir CRY stabilizörü olan TW68'in tip 2 diyabet tedavisinde terapötik potansiyelinin olduğunu göstermektedir.
Vibrio cholerae canlısında 6-4 fotoliyazının tespiti ve karakterize edilmesi
Ultra-violet (UV) light introduces mutagenic damages to the DNA in the form of pyrimidine dimers. If not repaired, these damages may cause carcinogenic and lethal effects on the organism. Photolyases (PHRs) are the ancient flavoproteins that repair UV-induced lesions in a blue light-dependent mechanism. PHRs, according to substrate specificity, are divided into two functional groups: CPD PHRs that repair cyclobutane pyrimidine dimers (CPD) and (6-4) PHRs that repair pyrimidine-pyrimidone (6-4) photoproducts [(6-4) PP]. Although CPD photolyases are distributed among the three domains of life, (6-4) PHRs were thought to be restricted to eukaryotes. Vibrio cholerae (O1 bivar Tor str. N16961) possesses three cryptochrome/photolyase family (CPF) members: VcPhr, a CPD photolyase; and two single-strand CPD PHRs VcCry1 and VcCry2. This dissertation presents the first (6-4) PHR in V. cholerae, named Vc (6-4) FeS-BCP. The enzyme belongs to newly discovered 'iron-sulphur cluster containing bacterial cryptochromes and photolyases' (FeS-BCP) class. Vc (6-4) FeS-BCP is the third enzyme characterized in this class. (6-4) repair activity of the Vc (6-4) FeS-BCP was demonstrated in vitro by DNA slot-blot repair assay. In vivo contribution of the protein to photoreactivation of the organism was presented by photoreactivation-complementation assay. Structural characterization of the protein via computational and experimental methods suggest that protein possesses FAD catalytic cofactor, DMRL photoantenna and [4FeS-4S] cluster.
Characterization of the role of CITED2 in circadian clock mechanism
Circadian rhythm is biochemical internal clock controlling physiological, behavioral and biochemical activities in a broad array of organisms from cyanobacteria to human. Mammalian circadian clock oscillates with 24 hours of period and environmental factors contribute to the entrainment of the rhythm. Many physiological variables are under the control of circadian clock in mammals such as sleep-wake cycles, body temperature, immune responses, cognitive functions, and hormone secretion. At the cellular level, similar clock mechanisms are found to be conserved among species. In mammals, molecular clock is composed of interlocked transcriptional-translational feedback loops (TTFL) which are controlled by positive and negative regulations. Positive regulation is modulated by the activity of BMAL1 and CLOCK so that BMAL1/CLOCK complex activates the transcription of CRYs and PERs. CRYs and PERs translocate into the nucleus and inhibit the transactivation of BMAL1/CLOCK thus CRY and PER constitute the negative arm of the molecular clock. In the last 20 years, genetics studies revealed that more proteins take part in the molecular clock mechanism. Previous high-throughput screening of transcription factor library identified novel molecular clock regulators that have the potential to affect BMAL1/CLOCK activity on E-box. CITED2 is one of the top candidates of this study that modulates BMAL1/CLOCK transactivation on Per1 promoter. Here in this thesis study, I aimed to characterize the role of CITED2 in the molecular clock mechanism at the cellular level. Initially, I showed that CITED2 has a repressor activity on BMAL1/CLOCK driven transcription on E-box element in a dose dependent manner. Knocking down of CITED2 altered the circadian phenotype by increasing circadian period by 1.5 hours and resulted in high levels of DBP and PER2 mRNA expression levels in U2-OS cell line. Affirmatively, CITED2 over expression lengthened the period and enhanced the amplitude of circadian rhythm in U2-OS cell line. At the molecular level, CITED2 over expression affected the transcriptional regulation and caused a reduction in DBP and PER2 levels. Finally, I showed that CITED2 reduces the physical interaction between BMAL1/CLOCK and it directly interacts with CLOCK. For the first time in this thesis, I presented evidence that CITED2 acts as a negative transcription factor on BMAL1/CLOCK driven transcription and it is a component of the circadian molecular clock. Prospectively, these findings will be beneficial for a better understanding of the link between the circadian clock and cellular activities.