RESUMO
RNA-binding proteins participate in diverse cellular processes, including DNA repair, post-transcriptional modification, and cancer progression through their interactions with RNAs, making them attractive for biotechnological applications. While nature provides an array of naturally occurring RNA-binding proteins, developing de novo RNA-binding peptides remains challenging. In particular, tailoring peptides to target single-stranded RNA with low complexity is difficult due to the inherent structural flexibility of RNA molecules. Here, we developed a codon-restricted mRNA display and identified multiple de novo peptides from a peptide library that bind to poly(C) and poly(A) RNA with KDs ranging from micromolar to submicromolar concentrations. One of the newly identified peptides is capable of binding to the cytosine-rich sequences of the oncogenic Cdk6 3'UTR RNA and MYU lncRNA, with affinity comparable to that of the endogenous binding protein. Hence, we present a novel platform for discovering de novo single-stranded RNA-binding peptides that offer promising avenues for regulating RNA functions.
Assuntos
Peptídeos , RNA , RNA Mensageiro/química , Peptídeos/química , Códon , Proteínas de Ligação a RNA/genéticaRESUMO
The reconstructed in vitro translation system known as the PURE system has been used in a variety of cell-free experiments such as the expression of native and de novo proteins as well as various display methods to select for functional polypeptides. We developed a refined PURE-based display method for the preparation of stable messenger RNA (mRNA) and complementary DNA (cDNA)-peptide conjugates and validated its utility for in vitro selection. Our conjugate formation efficiency exceeded 40%, followed by gel purification to allow minimum carry-over of components from the translation system to the downstream assay enabling clean and efficient random peptide sequence screening. We chose the commercially available anti-FLAG M2 antibody as a target molecule for validation. Starting from approximately 1.7 × 1012 random sequences, a round-by-round high-throughput sequencing showed clear enrichment of the FLAG epitope DYKDDD as well as revealing consensus FLAG epitope motif DYK(D/L/N)(L/Y/D/N/F)D. Enrichment of core FLAG motifs lacking one of the four key residues (DYKxxD) indicates that Tyr (Y) and Lys (K) appear as the two key residues essential for binding. Furthermore, the comparison between mRNA display and cDNA display method resulted in overall similar performance with slightly higher enrichment for mRNA display. We also show that gel purification steps in the refined PURE-based display method improve conjugate formation efficiency and enhance the enrichment rate of FLAG epitope motifs in later rounds of selection especially for mRNA display. Overall, the generalized procedure and consistent performance of two different display methods achieved by the commercially available PURE system will be useful for future studies to explore the sequence and functional space of diverse polypeptides.
Assuntos
DNA Complementar/genética , Epitopos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Biblioteca de Peptídeos , RNA Mensageiro/genética , HumanosRESUMO
Fe-S clusters are essential cofactors mediating electron transfer in respiratory and metabolic networks. However, obtaining active [4Fe-4S] proteins with heterologous expression is challenging due to (i) the requirements for [4Fe-4S] cluster assembly, (ii) the O2 lability of [4Fe-4S] clusters, and (iii) copurification of undesired proteins (e.g., ferredoxins). Here, we established a facile and efficient protocol to express mature [4Fe-4S] proteins in the PURE system under aerobic conditions. An enzyme aconitase and thermophilic ferredoxin were selected as model [4Fe-4S] proteins for functional verification. We first reconstituted the SUF system in vitro via a stepwise manner using the recombinant SUF subunits (SufABCDSE) individually purified from E. coli. Later, the incorporation of recombinant SUF helper proteins into the PURE system enabled mRNA translation-coupled [4Fe-4S] cluster assembly under the O2-depleted conditions. To overcome the O2 lability of [4Fe-4S] Fe-S clusters, an O2-scavenging enzyme cascade was incorporated, which begins with formate oxidation by formate dehydrogenase for NADH regeneration. Later, NADH is consumed by flavin reductase for FADH2 regeneration. Finally, bifunctional flavin reductase, along with catalase, removes O2 from the reaction while supplying FADH2 to the SufBC2D complex. These amendments enabled a one-pot, two-step synthesis of mature [4Fe-4S] proteins under aerobic conditions, yielding holo-aconitase with a maximum concentration of â¼0.15 mg/mL. This renovated system greatly expands the potential of the PURE system, paving the way for the future reconstruction of redox-active synthetic cells and enhanced cell-free biocatalysis.
Assuntos
Proteínas Ferro-Enxofre , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Escherichia coli/metabolismo , NAD/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Aconitato Hidratase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Flavinas/metabolismoRESUMO
3-(4-hydroxy-3-methoxyphenyl) propionic acid (HMPA) is a metabolite produced by the gut microbiota through the conversion of 4-hydroxy-3-methoxycinnamic acid (HMCA), which is a widely distributed hydroxycinnamic acid-derived metabolite found abundantly in plants. Several beneficial effects of HMPA have been suggested, such as antidiabetic properties, anticancer activities, and cognitive function improvement, in animal models and human studies. However, the intricate molecular mechanisms underlying the bioaccessibility and bioavailability profile following HMPA intake and the substantial modulation of metabolic homeostasis by HMPA require further elucidation. In this study, we effectively identified and characterized HMPA-specific GPR41 receptor, with greater affinity than HMCA. The activation of this receptor plays a crucial role in the anti-obesity effects and improvement of hepatic steatosis by stimulating the lipid catabolism pathway. For the improvement of metabolic disorders, our results provide insights into the development of functional foods, including HMPA, and preventive pharmaceuticals targeting GPR41.
Assuntos
Hempa , Metabolismo dos Lipídeos , Animais , Humanos , Hempa/metabolismo , Fígado/metabolismo , Propionatos/farmacologia , Propionatos/metabolismoRESUMO
One goal of origins of life research is to understand how primitive informational and catalytic biopolymers emerged and evolved. Recently, a number of sequencing techniques have been applied to analysis of replicating and evolving primitive biopolymer systems, providing a sequence-specific and high-resolution view of primitive chemical processes. Here, we review application of sequencing techniques to analysis of synthetic and primitive nucleic acids and polypeptides. This includes next-generation sequencing of primitive polymerization and evolution processes, followed by discussion of other novel biochemical techniques that could contribute to sequence analysis of primitive biopolymer driven chemical systems. Further application of sequencing to origins of life research, perhaps as a life detection technology, could provide insight into the origin and evolution of informational and catalytic biopolymers on early Earth or elsewhere.
RESUMO
Macroautophagy/autophagy plays a critical role in the pathogenesis of various human diseases including neurodegenerative disorders such as Parkinson disease (PD) and Huntington disease (HD). Chemical autophagy inducers are expected to serve as disease-modifying agents by eliminating cytotoxic/damaged proteins. Although many autophagy inducers have been identified, their precise molecular mechanisms are not fully understood because of the complicated crosstalk among signaling pathways. To address this issue, we performed several chemical genomic analyses enabling us to comprehend the dominancy among the autophagy-associated pathways followed by an aggresome-clearance assay. In a first step, more than 400 target-established small molecules were assessed for their ability to activate autophagic flux in neuronal PC12D cells, and we identified 39 compounds as autophagy inducers. We then profiled the autophagy inducers by testing their effect on the induction of autophagy by 200 well-established signal transduction modulators. Our principal component analysis (PCA) and clustering analysis using a dataset of "autophagy profiles" revealed that two Food and Drug Administration (FDA)-approved drugs, memantine and clemastine, activate endoplasmic reticulum (ER) stress responses, which could lead to autophagy induction. We also confirmed that SMK-17, a recently identified autophagy inducer, induced autophagy via the PRKC/PKC-TFEB pathway, as had been predicted from PCA. Finally, we showed that almost all of the autophagy inducers tested in this present work significantly enhanced the clearance of the protein aggregates observed in cellular models of PD and HD. These results, with the combined approach, suggested that autophagy-activating small molecules may improve proteinopathies by eliminating nonfunctional protein aggregates.Abbreviations: ADK: adenosine kinase; AMPK: AMP-activated protein kinase; ATF4: activating transcription factor 4; BECN1: beclin-1; DDIT3/CHOP: DNA damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FDA: Food and Drug Administration; GSH: glutathione; HD: Huntington disease; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; JAK: Janus kinase, MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K/MEK: mitogen-activated protein kinase kinase; MAP3K8/Tpl2: mitogen-activated protein kinase kinase kinase 8; MAPK: mitogen-activated protein kinase; MPP+: 1-methyl-4-phenylpyridinium; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; NAC: N-acetylcysteine; NGF: nerve growth factor 2; NMDA: N-methyl-D-aspartate; PCA: principal component analysis; PD: Parkinson disease; PDA: pancreatic ductal adenocarcinoma; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PMA: phorbol 12-myristate 13-acetate; PRKC/PKC: protein kinase C; ROCK: Rho-associated coiled-coil protein kinase; RR: ribonucleotide reductase; SIGMAR1: sigma non-opioid intracellular receptor 1; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: Transcription factor EB; TGFB/TGF-ß: Transforming growth factor beta; ULK1: unc-51 like autophagy activating kinase 1; XBP1: X-box binding protein 1.