ABSTRACT
During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as "Ago protein Affinity Purification by Peptides" (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.
Subject(s)
Argonaute Proteins/isolation & purification , Chromatography, Affinity/methods , Multiprotein Complexes/isolation & purification , Peptides/isolation & purification , Amino Acid Sequence , Animals , Cell Extracts , Chemical Precipitation , Drosophila melanogaster , Gene Silencing , HEK293 Cells , HeLa Cells , Humans , MicroRNAs/metabolism , Molecular Sequence Data , Peptides/chemistryABSTRACT
MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-ß pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago - TNRC6 levels.
Subject(s)
Argonaute Proteins/metabolism , Cell Nucleus/metabolism , RNA-Binding Proteins/metabolism , beta Karyopherins/metabolism , Active Transport, Cell Nucleus , Animals , Autoantigens/metabolism , Cell Line , Cytoplasm/metabolism , Gene Silencing , Humans , MiceABSTRACT
MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to target mRNAs, leading to gene silencing. However, Ago proteins are not the actual mediators of gene silencing but interact with a member of the GW182 protein family (also known as GW proteins), which coordinates all downstream steps in gene silencing. GW proteins contain an N-terminal Ago-binding domain that is characterized by multiple GW repeats and a C-terminal silencing domain with several globular domains. Within the Ago-binding domain, Trp residues mediate the direct interaction with the Ago protein. Here, we have characterized the interaction of Ago proteins with GW proteins in molecular detail. Using biochemical and NMR experiments, we show that only a subset of Trp residues engage in Ago interactions. The Trp residues are located in intrinsically disordered regions, where flanking residues mediate additional weak interactions, that might explain the importance of specific tryptophans. Using cross-linking followed by mass spectrometry, we map the GW protein interactions with Ago2, which allows for structural modeling of Ago-GW182 interaction. Our data further indicate that the Ago-GW protein interaction might be a two-step process involving the sequential binding of two tryptophans separated by a spacer with a minimal length of 10 aa.
Subject(s)
Argonaute Proteins/chemistry , Autoantigens/chemistry , Gene Expression Regulation/genetics , Models, Molecular , Multiprotein Complexes/chemistry , Protein Conformation , RNA-Binding Proteins/chemistry , Argonaute Proteins/metabolism , Autoantigens/metabolism , Baculoviridae , Circular Dichroism , Fluorescence Polarization , Genetic Vectors , HEK293 Cells , Humans , Immunoprecipitation , Magnetic Resonance Spectroscopy , Mass Spectrometry , Multiprotein Complexes/metabolism , Protein Binding , RNA Interference , RNA-Binding Proteins/metabolismABSTRACT
Argonaute proteins interact with small RNAs and facilitate small RNA-guided gene-silencing processes. Small RNAs guide Argonaute proteins to distinct target sites on mRNAs where Argonaute proteins interact with members of the GW182 protein family (also known as GW proteins). In subsequent steps, GW182 proteins mediate the downstream steps of gene silencing. The present mini-review summarizes and discusses our current knowledge of the molecular basis of Argonaute-GW182 protein interactions.
Subject(s)
Argonaute Proteins/physiology , Gene Silencing , RNA/metabolism , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Protein BindingABSTRACT
Polysorbates and Poloxamer 188 constitute the most common surfactants used in biopharmaceutical formulations owing to their excellent protein-stabilizing properties and good safety profiles. In recent years, however, a vast number of reports concerning potential risk factors closely related with their applications, such as the accumulation of degradation products, their inherent heterogeneity and adsorption effects of proteins at silicon/oil interfaces have drawn the focus to potential alternatives. Apart from tedious efforts to evaluate new excipient candidates, the use of mixed formulations leveraging combinations of well-established surfactants appears to be a promising approach to eliminate or, at least, minimize and postpone adverse effects associated with the single compounds. Due to the similar molecular properties of non-ionic surfactants, however, baseline separation of these mixtures, which is mandatory for their reliable quantification, poses a great challenge to analytical scientists. For this purpose, the present work describes the development of a robust mixed-mode liquid chromatography method coupled to evaporative light scattering detection (mixed-mode LC-ELSD) for simultaneous determination of the (intact) Polysorbate 20 and Poloxamer 188 content in biopharmaceutical formulations containing monoclonal antibodies. Extensive qualification and validation studies, comprising the evaluation of method specificity, robustness, linearity, accuracy and precision according to ICH guidelines, demonstrated its suitability for quality control studies. A case study on the storage stability of a formulated antibody was conducted to underline the method's practical utility. Finally, the versatility of the developed approach was successfully tested by quantifying Polysorbate 20-related surfactants, such as Polysorbate 80 and super-refined Polysorbate.
Subject(s)
Biological Products , Polysorbates , Chromatography, High Pressure Liquid , Poloxamer , Scattering, Radiation , Surface-Active AgentsABSTRACT
Argonaute proteins interact with small RNAs that guide them to complementary target RNAs, thus leading to inhibition of gene expression. Some but not all Argonaute proteins are endonucleases and can cleave the complementary target RNA. Here, we have mutated inactive human Ago1 and Ago3 and generated catalytic Argonaute proteins. We find that two short sequence elements at the N terminus are important for activity. In addition, PIWI-domain mutations in Ago1 may misarrange the catalytic center. Our work helps in understanding of the structural requirements that make an Argonaute protein an active endonucleolytic enzyme.