RESUMEN
Hepatic ischemia-reperfusion injury (IRI) is a major cause of liver damage during hepatic resection, transplantation, and other surgical procedures, often leading to graft failure and liver dysfunction. Recent studies have identified ferroptosis, a form of regulated cell death characterized by iron-dependent lipid peroxidation, as a key contributor to IRI. In this study, we investigated the protective effects of Ticlopidine, a thienopyridine compound and platelet aggregation inhibitor, on hepatic IRI. Using a C57BL/6J mouse model, we demonstrated that prophylactic Ticlopidine treatment significantly reduced necrotic and fibrotic areas in liver tissues, as well as serum levels of alanine transaminase (ALT) and aspartate aminotransferase (AST). Prussian Blue staining revealed that Ticlopidine pretreatment decreased iron accumulation in hepatic tissues, whereas markers of lipid peroxidation (malondialdehyde and 4-hydroxynonenal) and ferroptosis (PTGS2) were significantly downregulated. Additionally, Ticlopidine ameliorated inflammatory infiltration as indicated by reduced Gr-1 staining. In vitro, Ticlopidine dose-dependently inhibited ferroptosis induced by various inducers in liver cancer cell lines HUH7 and fibrosarcoma cells HT1080. The protective effects involved partial rescue of lipid peroxidation, significant reduction of ferrous iron levels, and strong protection against mitochondrial damage. These findings suggested that Ticlopidine acts as a broad-spectrum ferroptosis inhibitor, offering a promising therapeutic approach for protecting the liver against IRI.
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Telomeric repeat-binding factor 1 (Tbf1) has a similar architecture as the TRF family of telomeric proteins and plays important roles in both telomere homeostasis and ribosome regulation. However, the molecular basis of why Tbf1 has such different functions compared to other TRFs remains unclear. Here, we present the crystal structures of the TRF homology (TRFH) and Myb-L domains from Schizosaccharomyces pombe Tbf1 (spTbf1). TRFH-mediated homodimerization is essential for spTbf1 stability. Importantly, spTbf1TRFH lacks the conserved docking motif for interactions with telomeric proteins, explaining why spTbf1 does not participate in the assembly of the shelterin complex. Finally, structural and biochemical analyses demonstrate that TRFH and Myb-L domains as well as the loop region of spTbf1 coordinate to recognize S. pombe telomeric double-stranded DNA. Overall, our findings provide structural and functional insights into how fungi Tbf1 acts as an atypical telomeric repeat-binding factor, which helps to understand the evolution of TRFH-containing telomeric proteins.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Unión a Telómeros , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Unión al ADN , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/química , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Telómero/metabolismo , Telómero/química , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión a Telómeros/química , Factores de TranscripciónRESUMEN
OBJECTIVE: SUMO-specific protease 3 (SENP3), a member of the SUMO-specific protease family, reverses the SUMOylation of SUMO-2/3 conjugates. Dysregulation of SENP3 has been proven to be involved in the development of various tumors. However, its role in mantle cell lymphoma (MCL), a highly aggressive lymphoma, remains unclear. This study was aimed to elucidate the effect of SENP3 in MCL. METHODS: The expression of SENP3 in MCL cells and tissue samples was detected by RT-qPCR, Western blotting or immunohistochemistry. MCL cells with stable SENP3 knockdown were constructed using short hairpin RNAs. Cell proliferation was assessed by CCK-8 assay, and cell apoptosis was determined by flow cytometry. mRNA sequencing (mRNA-seq) was used to investigate the underlying mechanism of SENP3 knockdown on MCL development. A xenograft nude mouse model was established to evaluate the effect of SENP3 on MCL growth in vivo. RESULTS: SENP3 was upregulated in MCL patient samples and cells. Knockdown of SENP3 in MCL cells inhibited cell proliferation and promoted cell apoptosis. Meanwhile, the canonical Wnt signaling pathway and the expression of Wnt10a were suppressed after SENP3 knockdown. Furthermore, the growth of MCL cells in vivo was significantly inhibited after SENP3 knockdown in a xenograft nude mouse model. CONCLUSION: SENP3 participants in the development of MCL and may serve as a therapeutic target for MCL.
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Linfoma de Células del Manto , Adulto , Animales , Humanos , Ratones , Apoptosis/genética , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Modelos Animales de Enfermedad , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/patología , Ratones Desnudos , Proteínas del Tejido Nervioso , Péptido Hidrolasas/uso terapéutico , ARN Mensajero , Proteínas Wnt/uso terapéuticoRESUMEN
The conserved shelterin complex caps chromosome ends to protect telomeres and regulate telomere replication. In fission yeast Schizosaccharomyces pombe, shelterin consists of telomeric single- and double-stranded DNA-binding modules Pot1-Tpz1 and Taz1-Rap1 connected by Poz1, and a specific component Ccq1. While individual structures of the two DNA-binding OB folds of Pot1 (Pot1OB1-GGTTAC and Pot1OB2-GGTTACGGT) are available, structural insight into recognition of telomeric repeats with spacers by the complete DNA-binding domain (Pot1DBD) remains an open question. Moreover, structural information about the Tpz1-Ccq1 interaction requires to be revealed for understanding how the specific component Ccq1 of S. pombe shelterin is recruited to telomeres to function as an interacting hub. Here, we report the crystal structures of Pot1DBD-single-stranded-DNA, Pot1372-555-Tpz1185-212 and Tpz1425-470-Ccq1123-439 complexes and propose an integrated model depicting the assembly mechanism of the shelterin complex at telomeres. The structure of Pot1DBD-DNA unveils how Pot1 recognizes S. pombe degenerate telomeric sequences. Our analyses of Tpz1-Ccq1 reveal structural basis for the essential role of the Tpz1-Ccq1 interaction in telomere recruitment of Ccq1 that is required for telomere maintenance and telomeric heterochromatin formation. Overall, our findings provide valuable structural information regarding interactions within fission yeast shelterin complex at 3' ss telomeric overhang.
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Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Telomerasa , Proteínas Portadoras/genética , ADN de Cadena Simple , Unión Proteica , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Complejo Shelterina , Telomerasa/genética , Telómero/genética , Telómero/metabolismo , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
SARS-CoV-2 and its variants, such as the Omicron continue to threaten public health. The virus recognizes the host cell by attaching its Spike (S) receptor-binding domain (RBD) to the host receptor, ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here, we report the isolation and biological and structural characterization of a single-chain antibody (nanobody) from RBD-immunized alpaca. The nanobody, named DL28, binds to RBD tightly with a K D of 1.56 nM and neutralizes the original SARS-CoV-2 strain with an IC50 of 0.41 µg mL-1. Neutralization assays with a panel of variants of concern (VOCs) reveal its wide-spectrum activity with IC50 values ranging from 0.35 to 1.66 µg mL-1 for the Alpha/Beta/Gamma/Delta and an IC50 of 0.66 µg mL-1 for the currently prevalent Omicron. Competition binding assays show that DL28 blocks ACE2-binding. However, structural characterizations and mutagenesis suggest that unlike most antibodies, the blockage by DL28 does not involve direct competition or steric hindrance. Rather, DL28 may use a "conformation competition" mechanism where it excludes ACE2 by keeping an RBD loop in a conformation incompatible with ACE2-binding.
RESUMEN
SARS-CoV-2 engages with human cells through the binding of its Spike receptor-binding domain (S-RBD) to the receptor ACE2. Molecular blocking of this engagement represents a proven strategy to treat COVID-19. Here, we report a single-chain antibody (nanobody, DL4) isolated from immunized alpaca with picomolar affinity to RBD. DL4 neutralizes SARS-CoV-2 pseudoviruses with an IC50 of 0.101 µg mL-1 (6.2 nM). A crystal structure of the DL4-RBD complex at 1.75-Å resolution unveils the interaction detail and reveals a direct competition mechanism for DL4's ACE2-blocking and hence neutralizing activity. The structural information allows us to rationally design a mutant with higher potency. Our work adds diversity of neutralizing nanobodies against SARS-CoV-2 and should encourage protein engineering to improve antibody affinities in general.
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SARS-CoV-2 , Anticuerpos de Dominio Único , Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , Unión Proteica , Ingeniería de Proteínas , SARS-CoV-2/efectos de los fármacos , Anticuerpos de Dominio Único/farmacología , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
Telomeres, highly ordered DNA-protein complexes at eukaryotic linear chromosome ends, are specialized heterochromatin loci conserved among eukaryotes. In Schizosaccharomyces pombe, the shelterin complex is important for subtelomeric heterochromatin establishment. Despite shelterin has been demonstrated to mediate the recruitment of the Snf2/histone deacetylase-containing repressor complex (SHREC) and the Clr4 methyltransferase complex (CLRC) to telomeres, the mechanism involved in telomeric heterochromatin assembly remains elusive due to the multiple functions of the shelterin complex. Here, we found that CLRC plays a dominant role in heterochromatin establishment at telomeres. In addition, we identified a series of amino acids in the shelterin subunit Ccq1 that are important for the specific interaction between Ccq1 and the CLRC subunit Raf2. Finally, we demonstrated that the Ccq1-Raf2 interaction is essential for the recruitment of CLRC to telomeres, that contributes to histone H3 lysine 9 methylation, nucleosome stability and the shelterin-chromatin association, promoting a positive feedback mechanism for the nucleation and spreading of heterochromatin at subtelomeres. Together, our findings provide a mechanistic understanding of subtelomeric heterochromatin assembly by shelterin-dependent CLRC recruitment to chromosomal ends.
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Ensamble y Desensamble de Cromatina/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Heterocromatina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Metilación , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Complejo Shelterina/metabolismo , Complejo Shelterina/fisiología , Telómero/metabolismoRESUMEN
BACKGROUND: Analysis of viral protein-protein interactions is an essential step to uncover the viral protein functions and the molecular mechanism for the assembly of a viral protein complex. We employed a mammalian two-hybrid system to screen all the viral proteins of SARS-CoV-2 for the protein-protein interactions. RESULTS: Our study detected 48 interactions, 14 of which were firstly reported here. Unlike Nsp1 of SARS-CoV, Nsp1 of SARS-CoV-2 has the most interacting partners among all the viral proteins and likely functions as a hub for the viral proteins. Five self-interactions were confirmed, and five interactions, Nsp1/Nsp3.1, Nsp3.1/N, Nsp3.2/Nsp12, Nsp10/Nsp14, and Nsp10/Nsp16, were determined to be positive bidirectionally. Using the replicon reporter system of SARS-CoV-2, we screened all viral Nsps for their impacts on the viral replication and revealed Nsp3.1, the N-terminus of Nsp3, significantly inhibited the replicon reporter gene expression. We found Nsp3 interacted with N through its acidic region at N-terminus, while N interacted with Nsp3 through its NTD, which is rich in the basic amino acids. Furthermore, using purified truncated N and Nsp3 proteins, we determined the direct interactions between Nsp3 and N protein. CONCLUSIONS: Our findings provided a basis for understanding the functions of coronavirus proteins and supported the potential of interactions as the target for antiviral drug development.
RESUMEN
Serotonin N-acetyltransferase (SNAT) is the key rate-limiting enzyme in melatonin biosynthesis. It mediates melatonin biosynthesis in plants by using serotonin and 5-methoxytryptamine (5-MT), but little is known of its underlying mechanisms. Herein, we present a detailed reaction mechanism of a SNAT from Oryza sativa through combined structural and molecular dynamics (MD) analysis. We report the crystal structures of plant SNAT in the apo and binary/ternary complex forms with acetyl-CoA (AcCoA), serotonin, and 5-MT. OsSNAT exhibits a unique enzymatically active dimeric fold not found in the known structures of arylalkylamine N-acetyltransferase (AANAT) family. The key residues W188, D189, D226, N220, and Y233 located around the active pocket are important in catalysis, confirmed by site-directed mutagenesis. Combined with MD simulations, we hypothesize a novel plausible catalytic mechanism in which D226 and Y233 function as catalytic base and acid during the acetyl-transfer reaction.
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N-Acetiltransferasa de Arilalquilamina/química , Proteínas de Plantas/química , 5-Metoxitriptamina/química , 5-Metoxitriptamina/metabolismo , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , N-Acetiltransferasa de Arilalquilamina/genética , N-Acetiltransferasa de Arilalquilamina/metabolismo , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación , Oryza/enzimología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica , Serotonina/química , Serotonina/metabolismoRESUMEN
S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) are widely distributed among almost all organisms and often characterized with conserved Rossmann fold, TIM barrel, and D×G×G×G motif. However, some MTases show no methyltransferase activity. In the present study, the crystal structure of LepI, one MTase-like enzyme isolated from A. flavus that catalyzes pericyclic reactions, was investigated to determine its structure-function relationship. The overall structure of LepI in complex with the SAM mimic S-adenosyl-L-homocysteine (SAH) (PDB ID: 6IV7) indicated that LepI is a tetramer in solution. The residues His133, Arg197, Arg295, and Asp296 located near the active site can form hydrogen bonds with the substrate, thus participating in catalytic reactions. The binding of SAH in LepI is almost identical to that in other resolved MTases; however, the location of catalytic residues differs significantly. Phylogenetic trials suggest that LepI proteins share a common ancestor in plants and algae, which may explain the conserved SAM-binding site. However, the accelerated evolution of A. flavus has introduced both functional and structural changes in LepI. More importantly, the residue Arg295, which is unique to LepI, might be a key determinant for the altered enzymatic behavior. Collectively, the differences in the composition of catalytic residues, as well as the unique tetrameric form of LepI, define its unique enzymatic behavior. The present work provides an additional understanding of the structure-function relationship of MTases and MTase-like enzymes.
Asunto(s)
Aspergillus flavus/enzimología , Proteínas Fúngicas/química , Metiltransferasas/química , S-Adenosilhomocisteína/química , S-Adenosilmetionina/química , Secuencia de Aminoácidos , Aspergillus flavus/química , Aspergillus flavus/clasificación , Dominio Catalítico , Cristalografía por Rayos X , Evolución Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Enlace de Hidrógeno , Metiltransferasas/genética , Metiltransferasas/metabolismo , Modelos Moleculares , Filogenia , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Plant tryptophan decarboxylase (TDC) is a type II Pyridoxal-5'-phosphate-dependent decarboxylase (PLP_DC) that could be used as a target to genetically improve crops. However, lack of accurate structural information on plant TDC hampers the understanding of its decarboxylation mechanisms. In the present study, the crystal structures of Oryza sativa TDC (OsTDC) in its complexes with pyridoxal-5'-phosphate, tryptamine and serotonin were determined. The structures provide detailed interaction information between TDC and its substrates. The Y359 residue from the loop gate is a proton donor and forms a Lewis acid-base pair with serotonin/tryptamine, which is associated with product release. The H214 residue is responsible for PLP binding and proton transfer, and its proper interaction with Y359 is essential for OsTDC enzyme activity. The extra hydrogen bonds formed between the 5-hydroxyl group of serotonin and the backbone carboxyl groups of F104 and P105 explain the discrepancy between the catalytic activity of TDC in tryptophan and in 5-hydroxytryptophan. In addition, an evolutionary analysis revealed that type II PLP_DC originated from glutamic acid decarboxylase, potentially as an adaptive evolution of mechanism in organisms in extreme environments. This study is, to our knowledge, the first to present a detailed analysis of the crystal structure of OsTDC in these complexes. The information regarding the catalytic mechanism described here could facilitate the development of protocols to regulate melatonin levels and thereby contribute to crop improvement efforts to improve food security worldwide.
RESUMEN
Despite of important functions of strigolactones (SLs) and karrikins (KARs) in plant development, plant-parasite and plant-fungi interactions, their roles in soybean-rhizobia interaction remain elusive. SL/KAR signaling genes GmMAX2a, GmD14s, and GmKAIs are activated by rhizobia infection. GmMAX2a restored atmax2 root hair defects and soybean root hairs were changed in GmMAX2a overexpression (GmMAX2a-OE) or knockdown (GmMAX2a-KD) mutants. GmMAX2a-KD gave fewer, whereas GmMAX2a-OE produced more nodules than GUS hairy roots. Mutation of GmMAX2a in its KD or OE transgenic hairy roots affected the rhizobia infection-induced increases in early nodulation gene expression. Both mutant hairy roots also displayed the altered auxin, jasmonate and abscisic acid levels, as further verified by transcriptomic analyses of their synthetic genes. Overexpression of an auxin synthetic gene GmYUC2a also affected SL and KAR signaling genes. GmMAX2a physically interacted with SL/KAR receptors GmD14s, GmKAIs, and GmD14Ls with different binding affinities, depending on variations in the critical amino acids, forming active D14/KAI-SCFMAX2 complexes. The knockdown mutant roots of the nodule-specifically expressing GmKAIs and GmD14Ls gave fewer nodules, with altered expression of several early nodulation genes. The expression levels of GmKAIs, and GmD14Ls were markedly changed in GmMAX2a mutant roots, so did their target repressor genes GmD53s and GmSMAX1s. Thus, SL and KAR signaling were involved in soybean-rhizobia interaction and nodulation partly through interactions with hormones, and this may explain the different effects of MXA2 orthologs on legume determinate and indeterminate nodulation. The study provides fresh insights into the roles of GmMAX2-mediated SL/KAR signaling in soybean root hair and nodule formation.
Asunto(s)
Proteínas Portadoras/metabolismo , Furanos/metabolismo , Glycine max/metabolismo , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Lactonas/metabolismo , Proteínas de Plantas/metabolismo , Nodulación de la Raíz de la Planta/fisiología , Piranos/metabolismo , Transducción de Señal/fisiología , Bradyrhizobium , Proteínas Portadoras/genética , Regulación de la Expresión Génica de las Plantas , Técnicas de Silenciamiento del Gen , Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/genética , Nodulación de la Raíz de la Planta/genética , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Rhizobium , Transducción de Señal/genética , Glycine max/genética , TranscriptomaRESUMEN
Bergapten has long been used in combination with ultraviolet A irradiation to treat depigmentation disorder. However, extremely low bergapten contents in plants and difficulties in synthesizing bergapten have limited its application. Here, we developed an alternative bergapten-production method. We first determined the crystal structures of bergaptol O-methyltransferase from Peucedanum praeruptorum (PpBMT) and the ternary PpBMT-S-adenosyl-L-homocysteine (SAH)-bergaptol complex to identify key residues involved in bergaptol binding. Then, structure-based protein engineering was performed to obtain PpBMT mutants with improved catalytic activity towards bergaptol. Subsequently, a high-activity mutant was used to produce bergapten for pharmacological-activity analysis. Key PpBMT amino acids involved in bergaptol binding and substrate specificity were identified, such as Asp226, Asp246, Ser265, and Val320. Site-directed mutagenesis and biochemical analysis revealed that the V320I mutant efficiently transformed bergaptol to produce bergapten. Pharmacological-activity analysis indicated that bergapten positively affected hair pigmentation in mice and improved pigmentation levels in zebrafish embryos. This report provides the first description of the catalytic mechanism of coumarins-specific O-methyltransferase. The high-activity V320I mutant protein could be used in metabolic engineering to produce bergapten in order to treat depigmentation disorder. This structure-function study provides an alternative synthesis method and important advances for treating depigmentation disorders.