RESUMO
Ufmylation plays a crucial role in various cellular processes including DNA damage response, protein translation, and ER homeostasis. To date, little is known about how the enzymes responsible for ufmylation coordinate their action. Here, we study the details of UFL1 (E3) activity, its binding to UFC1 (E2), and its relation to UBA5 (E1), using a combination of structural modeling, X-ray crystallography, NMR, and biochemical assays. Guided by Alphafold2 models, we generate an active UFL1 fusion construct that includes its partner DDRGK1 and solve the crystal structure of this critical interaction. This fusion construct also unveiled the importance of the UFL1 N-terminal helix for binding to UFC1. The binding site suggested by our UFL1-UFC1 model reveals a conserved interface, and competition between UFL1 and UBA5 for binding to UFC1. This competition changes in the favor of UFL1 following UFM1 charging of UFC1. Altogether, our study reveals a novel, terminal helix-mediated regulatory mechanism, which coordinates the cascade of E1-E2-E3-mediated transfer of UFM1 to its substrate and provides new leads to target this modification.
Assuntos
Sítios de Ligação , Cristalografia por Raios XRESUMO
Ufmylation is a posttranslational modification in which the modifier UFM1 is attached to target proteins. This conjugation requires the concerted work of three enzymes named UBA5, UFC1, and UFL1. Initially, UBA5 activates UFM1 in a process that ends with UFM1 attached to UBA5's active site Cys. Then, in a trans-thiolation reaction, UFM1 is transferred from UBA5 to UFC1, forming a thioester bond with the latter. Finally, with the help of UFL1, UFM1 is transferred to the final destination-a lysine residue on a target protein. Therefore, not surprisingly, deletion of one of these enzymes abrogates the conjugation process. However, how overexpression of these enzymes affects this process is not yet clear. Here we found, unexpectedly, that overexpression of UBA5, but not UFC1, damages the ability of cells to migrate, in a similar way to cells lacking UBA5 or UFC1. At the mechanistic level, we found that overexpression of UBA5 reverses the trans-thiolation reaction, thereby leading to a back transfer of UFM1 from UFC1 to UBA5. This, as seen in cells lacking UBA5, reduces the level of charged UFC1 and therefore harms the conjugation process. In contrast, co-expression of UBA5 with UFM1 abolishes this effect, suggesting that the reverse transfer of UFM1 from UFC1 to UBA5 depends on the level of free UFM1. Overall, our results propose that the cellular expression level of the UFM1 conjugation enzymes has to be tightly regulated to ensure the proper directionality of UFM1 transfer.
Assuntos
Enzimas Ativadoras de Ubiquitina , Enzimas de Conjugação de Ubiquitina , Fenótipo , Processamento de Proteína Pós-Traducional , Proteínas/química , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Transfer RNAs (tRNAs) are fundamental molecules in cellular translation. In this study we have highlighted a fluorescence-based perceptive approach for tRNAs by using a quinoxaline small molecule. We have synthesised a water-soluble fluorescent pyrimido-quinoxaline-fused heterocycle containing a mandatory piperazine tail (DS1) with a large Stokes shift (â¼160â nm). The interaction between DS1 and tRNA results in significant fluorescence enhancement of the molecule with Kd â¼5â µM and multiple binding sites. Our work reveals that the DS1 binding site overlaps with the specific Mg2+ ion binding site in the D loop of tRNA. As a proof-of-concept, the molecule inhibited Pb2+ -induced cleavage of yeast tRNAPhe in the D loop. In competitive binding assays, the fluorescence of DS1-tRNA complex is quenched by a known tRNA-binder, tobramycin. This indicates the displacement of DS1 and, indeed, a substantiation of specific binding at the site of tertiary interaction in the central region of tRNA. The ability of compound DS1 to bind tRNA with a higher affinity compared to DNA and single-stranded RNA offers a promising approach to developing tRNA-based biomarker diagnostics in the future.
Assuntos
Compostos Heterocíclicos/química , Magnésio/química , Pirimidinas/química , Quinoxalinas/química , RNA de Transferência/química , Sítios de Ligação , Estrutura MolecularRESUMO
A winged bean trypsin inhibitor (WbTI-2) of molecular mass â¼20kDa, has been cloned and expressed in Escherichiacoli with full activity like the one from seed protein. It completely inhibits trypsin at an enzyme:inhibitor molar ratio of 1:2. PCR with cDNA and genomic DNA using same primers produced about 550 base pair product, which indicated it to be an intronless gene. Through site-directed mutagenesis, the Arg64 has been confirmed as the P1 residue. For the presence of five methionine residues in WbTI-2, cyanogen bromide (CNBr) digestion was carried out. Out of three fragments the one (about 65% of original size) containing the reactive site loop retained 50% activity.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Sequência de Bases , Clonagem Molecular , Brometo de Cianogênio/química , Fabaceae/metabolismo , Expressão Gênica , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Alinhamento de Sequência , Inibidores da Tripsina/biossíntese , Inibidores da Tripsina/genéticaRESUMO
Small molecules targeting G-quadruplex of oncogene promoter is considered as a promising anticancer therapeutics approach. Natural aloe compounds aloe emodin, and its glycoside derivative aloe emodin-8-glucoside and aloin have anticancer activity and also have potential DNA binding ability. These three compounds have promising binding ability towards quadruplex structures particularly c-KIT G-quadruplex. Here, this study demonstrates complete biophysical study of these compounds to c-KIT quadruplex structure. Aloe emodin showed highest binding stabilization with c-KIT which has been proved by absorbance, fluorescence, dye displacement, ITC and SPR studies. Moreover, comparative study of these compounds with HCT 116 cells line also agreed to their anti proliferative property which may be helpful to establish these aloe compounds as potential anticancer drugs. This study comprises a complete biophysical study along with their anti proliferative property and demonstrates aloe emodin as a potent c-KIT binding molecule.
Assuntos
Aloe , Quadruplex G , Aloe/química , Antraquinonas/farmacologiaRESUMO
Targeted intracellular delivery is an efficient strategy for developing therapeutics against cancer and other intracellular infections. Nonspecific drug delivery shows limited clinical applications owing to high dosage, cytotoxicity, nonspecific action, high cost, etc. Therefore, targeted delivery of less cytotoxic drug candidates to hepatocytes through ASGPR-mediated endocytosis could be an efficient strategy to surmount the prevailing shortcomings. In the present work, the gene encoding ASGPR-H1-CRD was amplified from Huh7 cells, cloned into pET 11a vector, and the ASGPR-H1-CRD protein was expressed and purified from E. coli. A novel triantennary galactose-conjugated quinoline derivative 4 was synthesized that demonstrates 17-fold higher binding affinity to isolated ASGPR-H1-CRD protein receptor (Kd â¼54â µM) in comparison to D-galactose (Kd â¼900â µM). Moreover, micro-calorimetric studies for the interaction of glycoconjugate 4 with ASGPR protein on live hepatocytes showed notable thermal response in case of ASGPR-containing Huh7 cells, in comparison to non-ASGPR Chang cells. These results might serve as an approach towards targeted delivery of small glycoconjugates to hepatocytes.
Assuntos
Receptor de Asialoglicoproteína/metabolismo , Glicoconjugados/farmacologia , Quinolinas/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Glicoconjugados/síntese química , Glicoconjugados/química , Humanos , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
Ufmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1's active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1's conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Domínio Catalítico/genética , Humanos , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/genética , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/isolamento & purificação , Enzimas Ativadoras de Ubiquitina/ultraestrutura , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/isolamento & purificação , Enzimas de Conjugação de Ubiquitina/ultraestrutura , Difração de Raios XRESUMO
Besides ubiquitin (Ub), humans have a set of ubiquitin-like proteins (UBLs) that can also covalently modify target proteins. To date, less is known about UBLs than Ub and even less is known about the UBL called ubiquitin-fold modifier 1 (UFM1). Currently, our understanding of protein modification by UFM1 (UFMylation) is like a jigsaw puzzle with many missing pieces, and in some cases it is not even clear whether these pieces of data are in the right place. Here we review the current data on UFM1 from structural biology to biochemistry and cell biology. We believe that the physiological significance of protein modification by UFM1 is currently underestimated and there is more to it than meets the eye.
Assuntos
Processamento de Proteína Pós-Traducional/fisiologia , Proteínas/metabolismo , Animais , Progressão da Doença , Humanos , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas/química , Ubiquitina/química , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/fisiologia , Enzimas de Conjugação de Ubiquitina/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitinação/fisiologiaRESUMO
Chitin deacetylase, an enzyme isolated from Cryptococcus laurentii RY1, catalyzes the hydrolysis of acetamido group of N-acetyl-D-glucosamine unit of chitin. The primary objective of this study was to characterize and comprehend the activation of chitin deacetylase by DMSO. The secondary structure of the protein was determined by circular dichroism(CD).The interaction of protein with DMSO was evaluated by CD and tryptophan fluorescence spectroscopy which revealed that DMSO had no effect on overall secondary structure, but induced change in the tertiary structure of the enzyme. The interaction of chitin deacetylase with chitin in DMSO system when investigated by molecular dynamics simulation revealed stronger chitin deacetylase-chitin interaction involving several amino acid residues. The enhanced activity of the enzyme in presence of DMSO along with the fact that its kcat is highest of all other reported chitin deacetylases makes it a superior candidate in the industrial sector involved in chitosan production from chitin.
RESUMO
Levansucrase is a secretary enzyme of Acetobacter nitrogenifigens strain RG1. The enzyme shows enhanced activity in the presence of Hg2+ in spite of being inhibited by other heavy metal ion Cd2+. In this study the structural characterization of levansucrase in native state as well as in the presence of Hg2+ and Cd2+ by CD spectroscopy is done. The secondary structures of the native enzyme and the enzyme treated with Hg2+ and Cd2+ on comparison by their CD spectra revealed that their spectra showed no significant difference indicating that both Hg2+ as well as Cd2+ had no effect on the overall secondary structure of the protein. The respective CD spectra on analysis revealed that they have almost identical percentage of secondary structural elements. The interaction of levansucrase with Hg2+ as well as Cd2+ was studied further by tryptophan fluorescence spectroscopy which on analysis revealed static quenching indicating protein-heavy metal complex formation. A blue shift in the tryptophan fluorescence spectra of Hg2+ treated protein indicated that the tryptophan residues have moved to a more hydrophobic environment in the protein away from aqueous phase. The mechanism of interaction of enzyme with mercury and cadmium was determined from their tryptophan fluorescence spectra.
Assuntos
Acetobacter/enzimologia , Proteínas de Bactérias/química , Cádmio/química , Hexosiltransferases/química , Mercúrio/química , Íons/química , Estrutura Secundária de ProteínaRESUMO
Protease inhibitors are essential bio-molecules that serve as a model system for the study of protein structure and protease-protease inhibitor interaction. We here report a bi-functional serine protease inhibitor from winged bean (WBCTI) that completely retains its inhibitory property against trypsin and chymotrypsin even after heating at 70°C. Detailed circular dichroism and fluorescence studies at different temperatures, 30-90°C, have been performed to understand the reason behind thermal stability of the protein. On the basis of our results it appears that WBCTI maintains its canonical structure up to 70°C. Above that the heat induced conformational change becomes irreversible which causes aggregation followed by precipitation of the protein. Moreover, the activity and stability of the secondary structure are found to decrease drastically in presence of dithiothreitol indicating that the protein acquires additional stability for the occurrence of two disulfide bonds. In addition to the structural characterization, an important property of WBCTI against the polyphagous pest Helicoverpa armigera has been explored in present study. WBCTI has showed reasonable inhibition of the mid-gut proteases of H. armigera. In artificial feeding trial through addition of WBCTI in diet resulted in significant growth retardation, delayed pupae formation and higher mortality of H. armigera larvae.