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Sirkadiyen ritim, siyanobakterilerden insanlara, bulunduğu organizmaların davranışsal, biyokimyasal ve fiziksel aktivitelerinin 24 saatte bir tekrarlanarak kontrol edilmesini sağlayan mekanizma olarak tanımlanır. Bu ritim içsel ve kendiliğinden olmasına rağmen çevresel ve sosyal faktörler ritmin fazını sirkadyen saat mekanizmasıyla 24 saate hassas olarak ayarlamaktadır. Memelilerde süprakiazmatik çekirdekte (SCN) bulunan ana saat bütün dokulardaki saatleri hormonlar ve sinir hücreleri sinyalleri ile senkronize etmektedir. Moleküler düzeyde saat mekanizması iç içe geçmiş iki adet transkripsiyon/translasyon geri bildirim döngüsü ile kontrol edilmektedir. Dört temel saat proteini saati idame ettirmektedir: bunlardan iki tanesi aktifleştirici (BMAL1 ve CLOCK) ve iki tanesi de baskılayıcıdır (PER ve CRY). BMAL1-CLCOK dimer oluşturarak, Cry ve Per genlerini de kapsayan protein kodlayan genlerin yaklaşık %40'ının promotör bölgesinde bulunan E-kutucuğuna bağlanır. PER ve CRY proteinleri sitoplazmada birikip çekirdeğe geçer ve CLOCK-BMAL1 ile etkileşir. Bu etkileşim zaman bağlı olarak ifade edilen genlerin ifadelerinin baskılanmasını sağlamakta ve bu durum 24 saatlik bir ritimle tekrarlanmaktadır. Hormon salınımı ve uyku-uyanma döngüsü gibi çeşitli biyolojik süreçler sirkadyen saat tarafından kontrol edilmektedir. Bu sebeple saatteki aksaklıklar depresyon, jet-lag, uyku bozukluğu, sinirsel hastalıklar, kalp-damar rahatsızlıkları, kanser, diyabet ve obezite gibi çeşitli hastalıklara yatkınlığı artırmaktadır. Ana saat proteinlerinden CRY, diyabet ve kanser ile ilişkilendirilmiştir. Yüksek CRY seviyesi karaciğerde glukagona bağlı glukoneogenezi baskılamakta iken, CRY'nin yokluğu kanserli farelerde apoptoza hassasiyeti artırmaktadır. Bundan dolayı CRY kararlılığını artıran ajanlar tip 2 diyabet tedavisi için kullanılabilirken, diğer taraftan CRY kararlılığını azaltan ajanlar kanser tedavisi için bir umut taşımaktadır. Bu çalışmada, yapıya bağlı ilaç tasarımı metodu ile CRY kararlılığını düzenleyen orijinal küçük moleküller keşfetmeyi hedefledik. İlk olarak fonksiyonu daha önceden belirlenmemiş ~2 milyon molekül, bilgisayar ortamında CRY'nin birinci ve ikinci çukurlarına yerleştirme metodu ile taranmıştır. Devamında, aday molekül sayısı hücre temelli sirkadyen ritim ve lusiferaz ile füzyon CRY1 proteininin yıkımı deneyleri gibi çeşitli biyokimyasal ve moleküler deneyler kullanılarak derece derece azaltılmıştır. En sonunda CRY1'e seçici olarak bağlanan ve onu kararsızlaştıran bir molekül (M47) tanımladık. M47, U2OS-Bmal1-dLuc hücrelerinde sirkadyen ritmin periyodunu uzatmış, genliğini azaltmış, CRY1 seviyesini düşürmüş ve sirkadyen gen çıktılarının ifadesini artırmıştır. İlave olarak, in vivo çalışmalarımız farelerin M47'nin belli dozlarını tolere edebildiğini ve M47'nin kanda 6 saat kaldığını, karaciğerde CRY1 seviyesini düşürürken Per2 ifadesini artırdığını göstermiştir. Çalışmamızda son olarak M47'nin p53-/- MEF hücrelerinde apoptozu artırdığını gösterdik. Farelerdeki toksisite ve farmakokinetik parametreler M47'nin P53 mutasyonuna bağlı olan kanser tedavilerinde ve diğer CRY1'e bağlı hastalıkların tedavilerinde kullanılabileceğini göstermiştir. The circadian rhythm controls the behavioral, biochemical and physical activities of the organisms in which exists from cyanobacteria to human. Although this rhythm is endogenous, environmental and social factors entrain the rhythm to align its phase exactly to 24h via circadian clock mechanism. In mammals, master clock located in the suprachiasmatic nuclei (SCN) synchronizes all clocks located in tissues via hormones and neuronal signals. The clock mechanism is controlled by two interconnected transcription/translation feedback loops (TTFL) at the molecular level. Four integral clock proteins drive the clock. These are two activators (BMAL1 and CLOCK) and two repressors (PER and CRY). BMAL1-CLOCK forms the dimer and binds to E-box in the promoter region of genes (~40% of all genes translated to proteins) including Cry and Per. PER and CRY proteins accumulate in the cytoplasm and translocate to the nucleus to inhibit BMAL1-CLOCK driven transactivation. Diverse biological processes like hormone secretion and sleep-wake cycles are controlled by the circadian clock. Therefore impairment of the clock increases the susceptibility to various diseases, such as depression, jetlag, sleep disorders, neurological and cardiovascular diseases, cancer, diabetes, and obesity. Among the core clock proteins, CRY is associated with diabetes and cancer. While high CRY abundance inhibits glucagon dependent gluconeogenesis in the liver, loss of CRY increases the susceptibility to apoptosis in cancer mice. Thus, while agents enhancing the stability of CRY can be utilized in the treatment of type2 diabetes; on the other hand agents diminishing the CRY stability can be a hope for cancer treatment. In this study, we aimed to discover novel small molecules regulating CRY stability by utilizing the structure-based drug design approach. Initially, ~2 million molecules with non-identified functions were docked to primary and secondary pockets of CRY1 in silico and then the number of selected molecules was gradually narrowed down by employing different biochemical and molecular assays such as by analyzing the effect of molecules on the cell-based circadian rhythm and degradation rate of CRY1 fused with luciferase. Finally, we identified a CRY1-binding small molecule (M47) that selectively destabilized CRY1. M47 increased period length and dampened the amplitude of the circadian rhythm, decreased the level of CRY1 and enhanced the circadian output gene expressions in U2OS Bmal1-dLuc cells. Further in vivo studies showed that mice could tolerate certain dosages of M47 where M47 had a half-life of 6h in blood and destabilized CRY1 and increased Per2 expression in mice liver. Finally, we showed that M47 enhanced apoptosis in p53-/- MEF cells. The toxicity and pharmacokinetic parameters from mice studies suggested that M47 may be used to improve the effectiveness of cancer treatment related to p53 mutations and other type of diseases related to CRY1. 154

Guanozin trifosfat (GTP)/guanozin difosfat (GDP) bağlayıcı G-proteinleri GTPaz aktiviteye sahip olup hücre düzenleyici ve sinyal gönderici potansiyelleri vardır. G-proteinleri GTP ye bağlı oldukları zaman aktif olup, GTP nin hidroliz yolu ile GDP ye dönüşmesinden sonra aktivitelerini yitirirler. G-proteinleri GTP ye bağlı kaldıkları süre boyunca sinyal iletmeye devam ederler. GTP ye bağlı G-proteinlerinin hidroliz mekanizması oldukça önem taşımasına rağmen ayrıntılı bir biçimde bilinmemektedir. GTP nin hidroliz mekanizmasının aydınlığa kavuşması spesifik aminoasitlerin bu tepkimedeki rollerini açıklayabilmek, (fazla/az) sinyal iletme esnasında oluşabilecek tümörlerin oluşumunu açıklayabilecek bilgilerin uzun vadede oluşmasında katkıda bulunacağından büyük önem taşımaktadır.Bulgularımız, Thermus aquaticus ve E.coli canlılarından gelen kristal yapılarında görülen Anahtar 1 bölgesinin, sulu ortama konduğunda çok hareketli ve kararsız bir yapısının olduğunu gösterdi. Henüz simülasyonlarımızın başında gözlemlediğimiz bu alandaki yapısal değişiklikler, Anahtar 1 bölgesinin ne kadar esnek olduğunu göstermektedir.Daha önceki çalışmalarda, özellikle de ribozom tarafından tetiklenen GTP hidroliz reaksiyonunda, His85 aminoasidinin saldıran suyu yönlendirerek geçiş konumuna yardımcı olduğu düşünülmekteydi. Çalışmamız His85 in etkinliği için iki adımın gerekli olduğunu gösterdi: Aktif bölgeye dönmek ve burada kararlı bir yapıya sahip olmak. Aşağıdaki faktörlerin His85 in aktif bölgeye dönmesine yardımcı olduğu düşünülmektedir:i)Protonlanma durumu, örneğin, His85 pozitif yüklü olduğu durumda yüksüz olduğu durumlarına göre daha çok aktif bölgede kalmak istediği belirlenmiştir.ii)Diğer aminoasitler aktif bölgede kalmasına yardımcı olabilir, örneğin, EF-Tu[D87E] mutasyona uğramış yapısından gelen simülasyonlarda, His85 in daha çok aktif bölgede kaldığı saptanmıştır.iii)Muhtemelen, ribozom His85 i aktif bölgeye getirip orada kararlı bir geometri kazanmasına yardımcı olmaktadır.Sonuçlarımız, His85 in aktif bölgede kararlı bir yapıya sahip olmasının, daha önceleri ?Su Sevmeyen Kapı? olarak adlandırılan Val20 ve Ile61 aminoasitleri tarafından sağlandığını göstermektedir. Bu iki aminoasit His85 in aktif bölgeye dönmesine engel oluşturduğu düşünülüyordu. Fakat bulgularımız bunların görevinin His85 e bir engel oluşturmak olmadığını, His85 aktif bölgeye dönünce onun imidazol halkasını bir kıskaç gibi tutarak dönmesine engel olup hidrolize yardımcı olmak olduğunu gösterdi. Bu kararlı yapı büyük ihtimalle ribozomun varlığında daha çok açığa çıkmaktadır. A large number of guanosine triphosphate (GTP)/ guanosine diphosphate (GDP) binding proteins (G-proteins), which usually have intrinsic (and/or stimulated) GTPase activity, are involved in a wide range of cellular regulatory and signal transduction processes. G-proteins generally exist in an active form when bound to GTP and become inactive when GTP is transformed to GDP through a hydrolysis reaction. The longer a GTP-binding protein remains in its active GTP-bound state, the longer it will transmit and amplify a certain signal. The mechanism of GTP hydrolysis is still largely unknown and of great importance to the understanding of the role of specific residues, which may lead to new and novel therapies for cancer.Experimental studies indicated the significance of some amino acid residues, e.g. His85, Asp87, Asp51, Thr62, Val20 and Ile61, for structural properties and GTPase activity of Elongation Factor-Tu (EF-Tu). In our study we aimed the comprehensive explanations of the roles of these amino acids.Our results indicated that, Switch 1 area is very unstable and dynamic when introduced into the solvent medium. Early conformational changes of this region in our simulations are showing the flexibility of the Switch 1 area. Thr62 was determined as the key factor for holding the position of Switch 1 region seen in the crystal structures coming from Thermus aquaticus and Escherichia coli species. On the other hand, Asp51 had no contribution to the position of this region.Previously, it was proposed that His85 stabilizes GTP hydrolysis transition state by orienting the attacking water molecule, especially in the ribosome induced mechanism. In our study two steps were determined for His85 activity: turning into the active site and to have a stable conformation in this site. We proposed that following factors can help His85 rotation into the active site:i)Protonation state of itself e.g. if it bears positive charge it has a tendency to stay in the active site more than the neutral form.ii)Other amino acids can help its stay in the active site e.g. His85 spent more time in the active site in the EF-Tu[D87E] mutant simulation.iii)Ribosome, probably, may bring and stabilize His85 into active site.Our findings suggested the stabilization of His85 in the active site could be achieved by Val20 and Ile61 which were previously called as the ?Hydrophobic Gate? residues. These residues were thought as the barrier for His85 rotation into the active site. However, our results indicated that the mission of Val20 and Ile61 is not forming a barrier for His85 rotation, but assisting the hydrolysis when it turns into the active site by holding the His85 imidazole ring, preventing its rotation, like a clamp. This stabilization probably mostly happens in the presence of ribosome. 87

Despite strict measures taken by many countries, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be an issue of global concern. Currently, there are no clinically proven pharmacotherapies for coronavirus disease 2019, despite promising initial results obtained from drugs such as azithromycin and hydroxychloroquine. Therefore, the repurposing of clinically approved drugs for use against SARS-CoV-2 has become a viable strategy. Here, we searched for drugs that target SARS-CoV-2 3C-like protease (3CL(pro)) and viral RNA-dependent RNA polymerase (RdRp) by in silico screening of the U.S. Food and Drug Administration approved drug library. Well-tolerated and widely used drugs were selected for molecular dynamics (MD) simulations to evaluate drug-protein interactions and their persistence under physiological conditions. Tetracycline, dihydroergotamine, ergotamine, dutasteride, nelfinavir, and paliperidone formed stable interactions with 3CL(pro)based on MD simulation results. Similar analysis with RdRp showed that eltrombopag, tipranavir, ergotamine, and conivaptan bound to the enzyme with high binding free energies. Docking results suggest that ergotamine, dihydroergotamine, bromocriptine, dutasteride, conivaptan, paliperidone, and tipranavir can bind to both enzymes with high affinity. As these drugs are well tolerated, cost-effective, and widely used, our study suggests that they could potentially to be used in clinical trials for the treatment of SARS-CoV-2-infected patients., NA

Attention deficit/hyperactivity disorder (ADHD) is a common and heritable phenotype frequently accompanied by insomnia, anxiety, and depression. Here, using a reverse phenotyping approach, we report heterozygous coding variations in the core circadian clock gene cryptochrome 1 in 15 unrelated multigenerational families with combined ADHD and insomnia. The variants led to functional alterations in the circadian molecular rhythms, providing a mechanistic link to the behavioral symptoms. One variant, CRY1Δ11 c.1657+3A>C, is present in approximately 1% of Europeans, therefore standing out as a diagnostic and therapeutic marker. We showed by exome sequencing in an independent cohort of patients with combined ADHD and insomnia that 8 of 62 patients and 0 of 369 controls carried CRY1Δ11. Also, we identified a variant, CRY1Δ6 c.825+1G>A, that shows reduced affinity for BMAL1/CLOCK and causes an arrhythmic phenotype. Genotype-phenotype correlation analysis revealed that this variant segregated with ADHD and delayed sleep phase disorder (DSPD) in the affected family. Finally, we found in a phenome-wide association study involving 9438 unrelated adult Europeans that CRY1Δ11 was associated with major depressive disorder, insomnia, and anxiety. These results defined a distinctive group of circadian psychiatric phenotypes that we propose to designate as "circiatric" disorders., NIH Clinical and Translational Science Award (CTSA) Program; NIH; French National Research Agency (ANR) under the “Investments for the future” Program; Integrative Biology of Emerging Infectious Diseases Laboratoire d’Excellence; IEIHSEER Grant; SEAe-Host Factors Grant; PNEUMOID Project Grant; INCA/Cancéropole Ile-de-France; Turkish Academy of Sciences (TÜBA); National Center for Advancing Translational Sciences (NCAST); Rockefeller University, INSERM; HHMI, University of Paris; St. Giles Foundation; Charles Bronfman Institute for Personalized Medicine at the Icahn School of Medicine at Mount Sinai

Cryptochromes are negative transcriptional regulators of the circadian clock in mammals. It is not clear how reducing the level of endogenous CRY1 in mammals will affect circadian rhythm and the relation of such a decrease with apoptosis. Here, we discovered a molecule (M47) that destabilizes Cryptochrome 1 (CRY1) both in vitro and in vivo. The M47 selectively enhanced the degradation rate of CRY1 by increasing its ubiquitination and resulted in increasing the circadian period length of U2OS Bmal1-dLuc cells. In addition, subcellular fractionation studies from mice liver indicated that M47 increased degradation of the CRY1 in the nucleus. Furthermore, M47-mediated CRY1 reduction enhanced oxaliplatin-induced apoptosis in Ras-transformed p53 null fibroblast cells. Systemic repetitive administration of M47 increased the median lifespan of p53(-/-) mice by similar to 25%. Collectively our data suggest that M47 is a promising molecule to treat forms of cancer depending on the p53 mutation., Scientific and Technological Research Council of Turkey (TÜBİTAK); Istanbul Development Agency

Human clock-gene variations contribute to the phenotypic differences observed in various behavioral and physiological processes, such as diurnal preference, sleep, metabolism, mood regulation, addiction, and fertility. However, little is known about the possible effects of identified variations at the molecular level. In this study, we performed a functional characterization at the cellular level of rare cryptochrome 2 (CRY2) missense variations that were identified from the Ensembl database. Our structural studies revealed that three variations (p.Pro123Leu, p.Asp406His, and p.Ser410Ile) are located at the rim of the secondary pocket of CRY2. We show that these variants were unable to repress CLOCK (circadian locomotor output cycles kaput)/BMAL1 (brain and muscle ARNT-like-1)-driven transcription in a cell-based reporter assay and had reduced affinity to CLOCK-BMAL1. Furthermore, our biochemical studies indicated that the variants were less stable than the WT CRY2, which could be rescued in the presence of period 2 (PER2), another core clock protein. Finally, we found that these variants were unable to properly localize to the nucleus and thereby were unable to rescue the circadian rhythm in a Cry1(-/-)Cry2(-/-) double KO mouse embryonic fibroblast cell line. Collectively, our data suggest that the rim of the secondary pocket of CRY2 plays a significant role in its nuclear localization independently of PER2 and in the intact circadian rhythm at the cellular level., Scientific and Technological Research Council of Turkey (TÜBİTAK); KBAG

Despite COVID-19 turned into a pandemic, no approved drug for the treatment or globally available vaccine is out yet. In such a global emergency, drug repurposing approach that bypasses a costly and long-time demanding drug discovery process is an effective way in search of finding drugs for the COVID-19 treatment. Recent studies showed that SARS-CoV-2 uses neuropilin-1 (NRP1) for host entry. Here we took advantage of structural information of the NRP1 in complex with C-terminal of spike (S) protein of SARSCoV-2 to identify drugs that may inhibit NRP1 and S protein interaction. U.S. Food and Drug Administration (FDA) approved drugs were screened using docking simulations. Among top drugs, well-tolerated drugs were selected for further analysis. Molecular dynamics (MD) simulations of drugs-NRP1 complexes were run for 100 ns to assess the persistency of binding. MM/GBSA calculations from MD simulations showed that eltrombopag, glimepiride, sitagliptin, dutasteride, and ergotamine stably and strongly bind to NRP1. In silico Alanine scanning analysis revealed that Tyr(297), Trp(301), and Tyr(353) amino acids of NRP1 are critical for drug binding. Validating the effect of drugs analyzed in this paper by experimental studies and clinical trials will expedite the drug discovery process for COVID-19., NA

Birt-Hogg-Dube syndrome (BHDS) is a rare disorder characterized by the triad of cutaneous lesions, renal tumors, lung cysts and inactivation of the gene folliculin (FLCN). Here, we present three female patients diagnosed with BHDS. First case a 55-year-old female had flesh moles histopathology compatible with angiofibroma, multiple cysts in the lung and kidneys, FLCN gene mutations ('c.1285dupC [p.His429Profs*]' 11th exon and 'c.653G>A [p.Arg258His]' 7th exon). The second case a 76-year-old female had trichodiscoma on her skin, multiple cysts in the lung, spontaneous pneumothorax, FLCN gene mutation 'c.1285dupC (p.His429Profs*27) 11th exon' and, her son had renal carcinoma history under 50 years of age. Our third case, also the daughter of case 2, had dermal papules histopathology compatible with trichodiscoma, spontaneous pneumothorax, FLCN gene mutation 'c.1285dupC (p.His429Profs*27) 11th exon' and, parotid oncocytoma. Through our cases, we document the first case of two mutations ('c.1285dupC [p.His429Profs*]' 11th exon and 'c.653G>A [p.Arg258His]' 7th exon) in the same FLCN gene and the 11th known case of parotid oncocytoma associated with BHDS in the light of the literature., NA

Mammalian circadian clocks are driven by transcription/ translation feedback loops composed of positive transcriptional activators (BMAL1 and CLOCK) and negative repressors (CRYPTOCHROMEs (CRYs) and PERIODs (PERs)). CRYs, in complex with PERs, bind to the BMAL1/CLOCK complex and repress E-box-driven transcription of clock-associated genes. There are two individual CRYs, with CRY1 exhibiting higher affinity to the BMAL1/CLOCK complex than CRY2. It is known that this differential binding is regulated by a dynamic serine-rich loop adjacent to the secondary pocket of both CRYs, but the underlying features controlling loop dynamics are not known. Here we report that allosteric regulation of the serine-rich loop is mediated by Arg-293 of CRY1, identified as a rare CRY1 SNP in the Ensembl and 1000 Genomes databases. The p.Arg293His CRY1 variant caused a shortened circadian period in a Cry1-/-Cry2-/-double knockout mouse embryonic fibroblast cell line. Moreover, the variant displayed reduced repressor activity on BMAL1/CLOCK driven transcription, which is explained by reduced affinity to BMAL1/ CLOCK in the absence of PER2 compared with CRY1.Molecular dynamics simulations revealed that the p.Arg293His CRY1 variant altered a communication pathway between Arg-293 and the serine loop by reducing its dynamicity. Collectively, this study provides direct evidence that allosterism in CRY1 is critical for the regulation of circadian rhythm., Scientific and Technological Research Council of Turkey (TÜBİTAK); Istanbul Development Agency Grant

Centriolar satellites are ubiquitous in vertebrate cells. They have recently emerged as key regulators of centrosome/cilium biogenesis, and their mutations are linked to ciliopathies. However, their precise functions and mechanisms of action remain poorly understood. Here, we generated a kidney epithelial cell line (IMCD3) lacking satellites by CRISPR/Cas9-mediated PCM1 deletion and investigated the cellular and molecular consequences of satellite loss. Cells lacking satellites still formed full-length cilia but at significantly lower numbers, with changes in the centrosomal and cellular levels of key ciliogenesis factors. Using these cells, we identified new ciliary functions of satellites such as regulation of ciliary content, Hedgehog signaling, and epithelial cell organization in three-dimensional cultures. However, other functions of satellites, namely proliferation, cell cycle progression, and centriole duplication, were unaffected in these cells. Quantitative transcriptomic and proteomic profiling revealed that loss of satellites affects transcription scarcely, but significantly alters the proteome. Importantly, the centrosome proteome mostly remains unaltered in the cells lacking satellites. Together, our findings identify centriolar satellites as regulators of efficient cilium assembly and function and provide insight into disease mechanisms of ciliopathies., European Research Council (ERC); European Union (European Union); Horizon 2020; EMBO Installation Grant; Newton Advanced Fellowship; Scientific and Technological Research Council of Turkey (TÜBİTAK); Science Academy of Turkey

Light is a very important environmental factor that governs many cellular responses in organisms. As a consequence, organisms possess different kinds of light-sensing photoreceptors to regulate their physiological variables and adapt to a given habitat. The cryptochrome/photolyase family (CPF) includes photoreceptors that perform different functions in different organisms. Photolyases repair ultraviolet-induced DNA damage by a process known as photoreactivation using photons absorbed from the blue end of the light spectrum. On the other hand, cryptochromes act as blue light circadian photoreceptors in plants and Drosophila to regulate growth and development. In mammals, cryptochromes have light-independent functions and are very important transcriptional regulators that act at the molecular level as negative transcriptional regulators of the circadian clock. In this review, we highlight current knowledge concerning the structural and functional relationships of CPF members., Scientific and Technological Research Council of Turkey (TÜBİTAK)