Nowe syntetyczne analogi kapu mRNA i ich zastosowania w badaniach biochemicznych i strukturalnych nad kompleksem enzymatycznym degradującym kap Dcp1/Dcp2 i innymi białkami oddziałującymi z kapem

Kap jest strukturą znajdującą się na 5’ końcu eukariotycznego mRNA mającą istotną rolę w metabolizmie komórki. Białka usuwające strukturę kapu takie jak kompleks Dcp1/2 mają natomiast znaczącą funkcję procesach degradacji mRNA. Jednak do tej pory ich mechanizm działania oraz sposoby regulacji aktywności nie zostały dokładnie poznane. Badania biochemiczne nad Dcp1/2 pokazały, że oddziałuje on specyficznie z m7GDP – produktem reakcji hydrolizy kapu. Zasugerowało to, że struktura m7GDP może być punktem wyjściowym dla syntezy nukleotydów mających większe powinowactwo do Dcp1/2, które mogą być wartościowymi narzędziami w badaniach biochemicznych. Przeprowadzono syntezy chemiczne oraz przebadano właściwości biologiczne serii analogów kapu opartych na strukturach nukleotydów takich jak m7GDP, m7GTP, oraz m7Gppppm7G. Związki te posiadają różnorodne modyfikacje w łańcuchu fosforanowym, takie jak grupa borano- lub tiofosforanowa, mające zwiększyć ich powinowactwo do białek wiążących kap. Otrzymano także szereg fluorescencyjnie znakowanych analogów kapu posiadające fluorofor o niewielkiej zawadzie sterycznej jako sondy molekularne w badaniach biofizycznych oraz narzędzia do syntezy fluorescencyjnego RNA. Po wykonaniu badań przesiewowych odkryto, że część z związków to inhibitory kompleksu Dcp1/2. Dalsze badania biochemiczne i strukturalne wykonano na najsilniejszym z inhibitorów (m7GpSpppSm7G izomer D3). Eksperymenty NMR pokazały położenie miejsca wiążącego inhibitor oraz powinowactwo tego związku do Dcp2. Badania kinetyczne pokazały, że badany inhibitor wiąże się zarówno z wolnym enzymem jak i kompleksem ES. Zaobserwowano też, że analogi kapu o wydłużonym łańcuchu fosforanowym są hydrolizowane przez Dcp2. Inne badania biochemiczne pokazały, że część z uzyskanych analogów kapu wykazuje silne powinowactwo do białka inicjującego translację eIF4E oraz posiadają obniżoną podatność na degradację enzymatyczną. Otrzymane fluorescencyjne analogi kapu są pierwszymi tego rodzaju związkami, które są inkorporowane do RNA in vitro. Niektóre z nich są także selektywnie odporne na działanie enzymów dekapujących Dcp1/2 bądź DcpS. Wstępne rezultaty badań biochemicznych świadczą, że znakowane fluorescencyjnie analogi kapu mogą być pomocne w badaniach oddziaływań kap-białko. W pracy opisano także przykładowe zastosowania modyfikowanych chemicznie analogów kapu w badania nad degradacją eukariotycznego mRNA. W ramach tych badań wykorzystano egzogenne mRNA zawierającego niehydrolizowany kap do śledzenia mechanizmów degradacji mRNA histonów w komórkach ssaczych. Dzięki temu poznano lepiej rolę Dcp1/2 w tym procesie oraz wpływie poliurydylacji 3’ końca mRNA na jego stabilność.The cap is a structure located on 5’ terminus of eukaryotic mRNA and it has a substantial role in cellular homeostasis. Hence proteins involved in cap removal, such as Dcp1/2 complex play a vital function in mRNA degradation. Nevertheless their mechanisms of both action and regulation remains obscure. Biochemical studies on Dcp2 protein have shown that Dcp2 interacts specifically with m7GDP (a product of the cap cleavage). It suggested that m7GDP structure could be a starting point for preparation of nucleotides displaying higher affinity to Dcp2, which could become valuable tools for biochemical research. A number of cap analogues based on m7GDP, m7GTP or m7Gppppm7G structures was synthesised and their biological properties were examined. These compounds possess a variety of chemical modifications in their phosphate chain, including tio- or boranophosphate groups, which are expected to increase their affinity to cap-interacting proteins. Furthermore, a set of fluorescently labelled cap analogues was also prepared. Since their fluorophores are very compact these nucleotides should be useful for preparation of fluorescent mRNA or as molecular probes. A screening assay revealed that some of prepared nucleotides are inhibitors of Dcp1/2. Further biochemical and structural researches were conducted using the most potent inhibitor (m7GpSpppSm7G, isomer D3). NMR experiments allowed to unravel the nucleotide binding site and its affinity to Dcp2. Kinetic studies showed that the inhibitor is bind by both free enzyme and ES complex. It also have been observed that cap analogues with elongated phosphate chain are hydrolysed by Dcp2. Other biochemical studies showed that some of prepared cap analogues display high affinity to eIF4 translation factor and lower susceptibility to enzymatic degradation. Fluorescent cap analogues obtained in this study are the first such compounds which can be incorporated into RNA in vitro. Some of them are also selectively resistant to either Dcp2 or DcpS decapping enzymes. Preliminary biochemical experiments indicated that these fluorescent cap analogues could be applied to studies on cap-protein interactions. Exemplary applications of chemically modified cap analogues to study mRNA degradation was also desribed in this study. These experiments were based on usage of egzogenous mRNA, containing a non-hydrolysable cap analogue, to monitor the degradation of histone mRNA in mammalian cells. Obtained results provided a better understanding of the role of Dcp2 in these processes and the influence of 3’ mRNA uridylation on its stability

Structure–Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin

Protein aggregation is a well-recognized problem in industrial preparation, including biotherapeutics. These low-energy states constantly compete with a native-like conformation, which is more pronounced in the case of macromolecules of low stability in the solution. A better understanding of the structure and function of such aggregates is generally required for the more rational development of therapeutic proteins, including single-chain fusion cytotoxins to target specific receptors on cancer cells. Here, we identified and purified such particles as side products of the renaturation process of the single-chain fusion cytotoxin, composed of two diphtheria toxin (DT) domains and interleukin 13 (IL-13), and applied various experimental techniques to comprehensively understand their molecular architecture and function. Importantly, we distinguished soluble purified dimeric and fractionated oligomeric particles from aggregates. The oligomers are polydisperse and multimodal, with a distribution favoring lower and even stoichiometries, suggesting they are composed of dimeric building units. Importantly, all these oligomeric particles and the monomer are cystine-dependent as their innate disulfide bonds have structural and functional roles. Their reduction triggers aggregation. Presumably the dimer and lower oligomers represent the metastable state, retaining the native disulfide bond. Although significantly reduced in contrast to the monomer, they preserve some fraction of bioactivity, manifested by their IL-13RA2 receptor affinity and selective cytotoxic potency towards the U-251 glioblastoma cell line. These molecular assemblies probably preserve structural integrity and native-like fold, at least to some extent. As our study demonstrated, the dimeric and oligomeric cytotoxin may be an exciting model protein, introducing a new understanding of its monomeric counterpart’s molecular characteristics

Structural basis of mRNA-cap recognition by Dcp1-Dcp2.

Removal of the 5' cap on mRNA by the decapping enzyme Dcp2 is a critical step in 5'-to-3' mRNA decay. Understanding the structural basis of Dcp2 activity has been a challenge because Dcp2 is dynamic and has weak affinity for the cap substrate. Here we present a 2.6-Å-resolution crystal structure of a heterotrimer of fission yeast Dcp2, its essential activator Dcp1, and the human NMD cofactor PNRC2, in complex with a tight-binding cap analog. Cap binding is accompanied by a conformational change in Dcp2, thereby forming a composite nucleotide-binding site comprising conserved residues in the catalytic and regulatory domains. Kinetic analysis of PNRC2 revealed that a conserved short linear motif enhances both substrate affinity and the catalytic step of decapping. These findings explain why Dcp2 requires a conformational change for efficient catalysis and reveals that coactivators promote RNA binding and the catalytic step of decapping, possibly through different conformational states

Structural basis of mRNA-cap recognition by Dcp1–Dcp2

Removal of the 5' cap on mRNA by the decapping enzyme Dcp2 is a critical step in 5'-to-3' mRNA decay. Understanding the structural basis of Dcp2 activity has been a challenge because Dcp2 is dynamic and has weak affinity for the cap substrate. Here we present a 2.6-Å-resolution crystal structure of a heterotrimer of fission yeast Dcp2, its essential activator Dcp1, and the human NMD cofactor PNRC2, in complex with a tight-binding cap analog. Cap binding is accompanied by a conformational change in Dcp2, thereby forming a composite nucleotide-binding site comprising conserved residues in the catalytic and regulatory domains. Kinetic analysis of PNRC2 revealed that a conserved short linear motif enhances both substrate affinity and the catalytic step of decapping. These findings explain why Dcp2 requires a conformational change for efficient catalysis and reveals that coactivators promote RNA binding and the catalytic step of decapping, possibly through different conformational states

Structure of the activated Edc1-Dcp1-Dcp2-Edc3 mRNA decapping complex with substrate analog poised for catalysis.

The conserved decapping enzyme Dcp2 recognizes and removes the 5' eukaryotic cap from mRNA transcripts in a critical step of many cellular RNA decay pathways. Dcp2 is a dynamic enzyme that functions in concert with the essential activator Dcp1 and a diverse set of coactivators to selectively and efficiently decap target mRNAs in the cell. Here we present a 2.84 Å crystal structure of K. lactis Dcp1-Dcp2 in complex with coactivators Edc1 and Edc3, and with substrate analog bound to the Dcp2 active site. Our structure shows how Dcp2 recognizes cap substrate in the catalytically active conformation of the enzyme, and how coactivator Edc1 forms a three-way interface that bridges the domains of Dcp2 to consolidate the active conformation. Kinetic data reveal Dcp2 has selectivity for the first transcribed nucleotide during the catalytic step. The heterotetrameric Edc1-Dcp1-Dcp2-Edc3 structure shows how coactivators Edc1 and Edc3 can act simultaneously to activate decapping catalysis

Structure of the activated Edc1-Dcp1-Dcp2-Edc3 mRNA decapping complex with substrate analog poised for catalysis.

The conserved decapping enzyme Dcp2 recognizes and removes the 5' eukaryotic cap from mRNA transcripts in a critical step of many cellular RNA decay pathways. Dcp2 is a dynamic enzyme that functions in concert with the essential activator Dcp1 and a diverse set of coactivators to selectively and efficiently decap target mRNAs in the cell. Here we present a 2.84 Å crystal structure of K. lactis Dcp1-Dcp2 in complex with coactivators Edc1 and Edc3, and with substrate analog bound to the Dcp2 active site. Our structure shows how Dcp2 recognizes cap substrate in the catalytically active conformation of the enzyme, and how coactivator Edc1 forms a three-way interface that bridges the domains of Dcp2 to consolidate the active conformation. Kinetic data reveal Dcp2 has selectivity for the first transcribed nucleotide during the catalytic step. The heterotetrameric Edc1-Dcp1-Dcp2-Edc3 structure shows how coactivators Edc1 and Edc3 can act simultaneously to activate decapping catalysis

Two-headed tetraphosphate cap analogs are inhibitors of the Dcp1/2 RNA decapping complex

Dcp1/2 is the major eukaryotic RNA decapping complex, comprised of the enzyme Dcp2 and activator Dcp1, which removes the 5′ m(7)G cap from mRNA, committing the transcript to degradation. Dcp1/2 activity is crucial for RNA quality control and turnover, and deregulation of these processes may lead to disease development. The molecular details of Dcp1/2 catalysis remain elusive, in part because both cap substrate (m(7)GpppN) and m(7)GDP product are bound by Dcp1/2 with weak (mM) affinity. In order to find inhibitors to use in elucidating the catalytic mechanism of Dcp2, we screened a small library of synthetic m(7)G nucleotides (cap analogs) bearing modifications in the oligophosphate chain. One of the most potent cap analogs, m(7)Gp(S)ppp(S)m(7)G, inhibited Dcp1/2 20 times more efficiently than m(7)GpppN or m(7)GDP. NMR experiments revealed that the compound interacts with specific surfaces of both regulatory and catalytic domains of Dcp2 with submillimolar affinities. Kinetics analysis revealed that m(7)Gp(S)ppp(S)m(7)G is a mixed inhibitor that competes for the Dcp2 active site with micromolar affinity. m(7)Gp(S)ppp(S)m(7)G-capped RNA undergoes rapid decapping, suggesting that the compound may act as a tightly bound cap mimic. Our identification of the first small molecule inhibitor of Dcp2 should be instrumental in future studies aimed at understanding the structural basis of RNA decapping and may provide insight toward the development of novel therapeutically relevant decapping inhibitors

Preparation of Synthetically Challenging Nucleotides Using Cyanoethyl P‑Imidazolides and Microwaves

We describe a general method for the elongation of nucleoside oligophosphate chains by means of cyanoethyl (CE) phosphorimidazolides. Though the method requires a phosphorylation and subsequent deprotection reaction, both steps could be achieved in one pot without isolation/purification of the initial phosphorylation product. We have also found that pyrophosphate bond formation by this method is significantly accelerated by microwave irradiation

Two-headed tetraphosphate cap analogs are inhibitors of the Dcp1/2 RNA decapping complex

Dcp1/2 is the major eukaryotic RNA decapping complex, comprised of the enzyme Dcp2 and activator Dcp1, which removes the 5' m(7)G cap from mRNA, committing the transcript to degradation. Dcp1/2 activity is crucial for RNA quality control and turnover, and deregulation of these processes may lead to disease development. The molecular details of Dcp1/2 catalysis remain elusive, in part because both cap substrate (m(7)GpppN) and m(7)GDP product are bound by Dcp1/2 with weak (mM) affinity. In order to find inhibitors to use in elucidating the catalytic mechanism of Dcp2, we screened a small library of synthetic m(7)G nucleotides (cap analogs) bearing modifications in the oligophosphate chain. One of the most potent cap analogs, m(7)GpSpppSm(7)G, inhibited Dcp1/2 20 times more efficiently than m(7)GpppN or m(7)GDP. NMR experiments revealed that the compound interacts with specific surfaces of both regulatory and catalytic domains of Dcp2 with submillimolar affinities. Kinetics analysis revealed that m(7)GpSpppSm(7)G is a mixed inhibitor that competes for the Dcp2 active site with micromolar affinity. m(7)GpSpppSm(7)G-capped RNA undergoes rapid decapping, suggesting that the compound may act as a tightly bound cap mimic. Our identification of the first small molecule inhibitor of Dcp2 should be instrumental in future studies aimed at understanding the structural basis of RNA decapping and may provide insight toward the development of novel therapeutically relevant decapping inhibitors