Mammalian Cell Extracts Research Articles

Use of qPCR to Evaluate Efficiency of the Bulky DNA Damage Removal in Extracts of Mammalian Cells with Different Maximum Lifespan

Proteins of nucleotide excision repair system (NER) are responsible for detecting and removing a wide range of bulky DNA damages, thereby contributing significantly to the genome stability maintenance within mammalian cells. Evaluation of NER functional status in the cells is important for identifying pathological changes in the body and assessing effectiveness of chemotherapy. The following method, described herein, has been developed for better assessment of bulky DNA damages removal in vitro, based on qPCR. Using the developed method, NER activity was compared for the extracts of the cells from two mammals with different lifespans: a long-lived naked mole-rat (Heterocephalus glaber) and a short-lived mouse (Mus musculus). Proteins of the H. glaber cell extract have been shown to be 1.5 times more effective at removing bulky damage from the model DNA substrate than the proteins of the M. musculus cell extract. These results are consistent with the experimental data previously obtained. The presented method could be applied not only in fundamental studies of DNA repair in mammalian cells, but also in clinical practice.

RNA-Binding Protein-Mediated mRNA Deadenylation in Mammalian Cell Extracts.

Removal of the poly(A) tail, or deadenylation, is a crucial step in destabilizing mRNAs in eukaryotes. In this chapter, we describe a cell-free deadenylation assay that uses cytoplasmic cell extracts from human HEK293 cells transiently transfected with DNA encoding RNA-binding proteins (RBP), and in vitro-transcribed, radiolabeled, RNA probes. We include methods to evaluate the effects of RBPs or deadenylases on various in vitro-transcribed probes, with or without poly(A) tails. Finally, we also demonstrate the adaptability of these assays to test purified protein components in our cell-free deadenylation assay. In our experience, these methods are well suited for the initial assessment of the effects of RBPs on the deadenylation of mRNAs.

Methods to Study Z-DNA-Induced Genetic Instability.

Alternative DNA structures that differ from the canonical B-DNA double helix, including Z-DNA, have received much attention recently due to their impact on DNA metabolic processes, including replication, transcription, and genome maintenance. Non-B-DNA-forming sequences can also stimulate genetic instability associated with disease development and evolution. Z-DNA can stimulate different types of genetic instability events in different species, and several different assays have been established to detect Z-DNA-induced DNA strand breaks and mutagenesis in prokaryotic andeukaryotic systems. In this chapter, we will introduce some of these methods including Z-DNA-induced mutation screening and detection of Z-DNA-induced strand breaks in mammalian cells, yeast, and mammalian cell extracts. Results from these assays should provide better insight into the mechanisms of Z-DNA-related genetic instability in different eukaryotic model systems.

Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin

BackgroundThe cohesin complex organizes the genome-forming dynamic chromatin loops that impact on all DNA-mediated processes. There are two different cohesin complexes in vertebrate somatic cells, carrying the STAG1 or STAG2 subunit, and two versions of the regulatory subunit PDS5, PDS5A and PDS5B. Mice deficient for any of the variant subunits are embryonic lethal, which indicates that they are not functionally redundant. However, their specific behavior at the molecular level is not fully understood.ResultsThe genome-wide distribution of cohesin provides important information with functional consequences. Here, we have characterized the distribution of cohesin subunits and regulators in mouse embryo fibroblasts (MEFs) either wild type or deficient for cohesin subunits and regulators by chromatin immunoprecipitation and deep sequencing. We identify non-CTCF cohesin-binding sites in addition to the commonly detected CTCF cohesin sites and show that cohesin-STAG2 is the preferred variant at these positions. Moreover, this complex has a more dynamic association with chromatin as judged by fluorescence recovery after photobleaching (FRAP), associates preferentially with WAPL and is more easily extracted from chromatin with salt than cohesin-STAG1. We observe that both PDS5A and PDS5B are exclusively located at cohesin-CTCF positions and that ablation of a single paralog has no noticeable consequences for cohesin distribution while double knocked out cells show decreased accumulation of cohesin at all its binding sites. With the exception of a fraction of cohesin positions in which we find binding of all regulators, including CTCF and WAPL, the presence of NIPBL and PDS5 is mutually exclusive, consistent with our immunoprecipitation analyses in mammalian cell extracts and previous results in yeast.ConclusionOur findings support the idea that non-CTCF cohesin-binding sites represent sites of cohesin loading or pausing and are preferentially occupied by the more dynamic cohesin-STAG2. PDS5 proteins redundantly contribute to arrest cohesin at CTCF sites, possibly by preventing binding of NIPBL, but are not essential for this arrest. These results add important insights towards understanding how cohesin regulates genome folding and the specific contributions of the different variants that coexist in the cell.

A Photo-Crosslinking Approach to Identify Class II SUMO-1 Binders.

The small ubiquitin-like modifier (SUMO) is involved in various cellular processes and mediates known non-covalent protein-protein interactions by three distinct binding surfaces, whose interactions are termed class I to class III. While interactors for the class I interaction, which involves binding of a SUMO-interacting motif (SIM) to a hydrophobic groove in SUMO-1 and SUMO-2/3, are widely abundant, only a couple of examples have been reported for the other two types of interactions. Class II binding is conveyed by the E67 loop region on SUMO-1. Many previous studies to identify SUMO binders using pull-down or microarray approaches did not strategize on the SUMO binding mode. Identification of SUMO binding partners is further complicated due to the typically transient and low affinity interactions with the modifier. Here we aimed to identify SUMO-1 binders selectively enriched for class II binding. Using a genetically encoded photo-crosslinker approach, we have designed SUMO-1 probes to covalently capture class II SUMO-1 interactors by strategically positioning the photo-crosslinking moiety on the SUMO-1 surface. The probes were validated using known class II and class I binding partners. We utilized the probe with p-benzoyl-phenylalanine (BzF, also termed BpF or Bpa) at the position of Gln69 to identify binding proteins from mammalian cell extracts using mass spectrometry. By comparison with results obtained with a similarly designed SUMO-1 probe to target SIM-mediated binders of the class I type, we identified 192 and 96 proteins specifically enriched by either probe, respectively. The implicated preferential class I or class II binding modes of these proteins will further contribute to unveiling the complex interplay of SUMO-1-mediated interactions.

Development of in vitro-grown spheroids as a 3D tumor model system for solid-state NMR spectroscopy

Recent advances in the field of in-cell NMR spectroscopy have made it possible to study proteins in the context of bacterial or mammalian cell extracts or even entire cells. As most mammalian cells are part of a multi-cellular complex, there is a need to develop novel NMR approaches enabling the study of proteins within the complexity of a 3D cellular environment. Here we investigate the use of the hanging drop method to grow spheroids which are homogenous in size and shape as a model system to study solid tumors using solid-state NMR (ssNMR) spectroscopy. We find that these spheroids are stable under magic-angle-spinning conditions and show a clear change in metabolic profile as compared to single cell preparations. Finally, we utilize dynamic nuclear polarization (DNP)-supported ssNMR measurements to show that low concentrations of labelled nanobodies targeting EGFR (7D12) can be detected inside the spheroids. These findings suggest that solid-state NMR can be used to directly examine proteins or other biomolecules in a 3D cellular microenvironment with potential applications in pharmacological research.

Nanoparticle-based local translation reveals mRNA as a translation-coupled scaffold with anchoring function

The spatial regulation of messenger RNA (mRNA) translation is central to cellular functions and relies on numerous complex processes. Biomimetic approaches could bypass these endogenous complex processes, improve our comprehension of the regulation, and allow for controlling local translation regulations and functions. However, the causality between local translation and nascent protein function remains elusive. Here, we developed a nanoparticle (NP)-based strategy to magnetically control mRNA spatial patterns in mammalian cell extracts and investigate how local translation impacts nascent protein localization and function. By monitoring the translation of the magnetically localized mRNAs, we show that mRNA-NP complexes operate as a source for the continuous production of proteins from defined positions. By applying this approach to actin-binding proteins, we triggered the local formation of actin cytoskeletons and identified the minimal requirements for spatial control of the actin filament network. In addition, our bottom-up approach identified a role for mRNA as a translation-coupled scaffold for the function of nascent N-terminal protein domains. Our approach will serve as a platform for regulating mRNA localization and investigating the function of nascent protein domains during translation.

Assay for β-Galactosidase in Extracts of Mammalian Cells.

This protocol contains a method for the detection of β-galactosidase expressed from reporter vectors that have been transfected into mammalian cells. The assay is both simple and rapid and can be performed using a visible light spectrophotometer.

A proof-of-concept study in engineering synthetic protein for selective recognition of substrate-free polyubiquitin.

Similar to substrate-conjugated polyubiquitin, unanchored polyubiquitin chains are emerging as important regulators for diverse biological processes. The affinity purification of unanchored polyubiquitin from various organisms has been reported, however, tools able to distinguish unanchored polyubiquitin chains with different isopeptide linkages have not yet been described. Toward the goal of selectively identifying and purifying unanchored polyubiquitin chains linked through different Lysines, Scott etal. developed a novel strategy in their study [Proteomics 2016, 16, 1961-1969]. They designed a linker-optimized ubiquitin-binding domain hybrid (t-UBD) containing two UBDs, a ZnFCUBP domain, and a linkage-selective UBA domain, to specifically recognize unanchored Lys48-linked polyubiquitin chains. Subsequently, a series of assays has proved the feasibility of this novel strategy for the purification of endogenous substrate-free Lys48-linked polyubiquitin chains from mammalian cell extracts. Their research not only provides a tool for purifying unanchored polyubiquitin with different isopeptide linkages, but also paves the way for generating reagents to study the function of unanchored polyubiquitin chains of different linkages in the future. The design of UBD hybrids for defined unanchored polyubiquitin (Lys48-polyubiquitin) in this study also set an excellent example for future methodology studies regarding monitoring in vivo dynamic changes in the patterns of ubiquitination.

Mass spectrometry insights into a tandem ubiquitin-binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin.

Unanchored polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate-conjugated polyubiquitin. However tools able to discriminate unanchored polyubiquitin chains of different isopeptide linkages have not been reported. We describe the design of a linker-optimized ubiquitin-binding domain hybrid (t-UBD) containing two UBDs, a ZnF-UBP domain in tandem with a linkage-selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48-linked polyubiquitin chains. Utilizing native MS to quantitatively probe binding affinities we confirm cooperative binding of the UBDs within the synthetic protein, and desired binding specificity for Lys48-linked ubiquitin dimers. Furthermore, MS/MS analyses indicate that the t-UBD, when applied as an affinity enrichment reagent, can be used to favor the purification of endogenous unanchored Lys48-linked polyubiquitin chains from mammalian cell extracts. Our study indicates that strategies for the rational design and engineering of polyubiquitin chain-selective binding in nonbiological polymers are possible, paving the way for the generation of reagents to probe unanchored polyubiquitin chains of different linkages and more broadly the 'ubiquitome'. All MS data have been deposited in the ProteomeXchange with identifier PXD004059 (http://proteomecentral.proteomexchange.org/dataset/PXD004059).

TOPORS, a Dual E3 Ubiquitin and Sumo1 Ligase, Interacts with 26 S Protease Regulatory Subunit 4, Encoded by the PSMC1 Gene.

The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been highlighted in the context of neurodegenerative diseases, including retinal dystrophies. TOPORS, a dual E3 ubiquitin and SUMO1 ligase, forms a component of the UPS and selected substrates for its enzymatic activities, such as DJ-1/PARK7 and APOBEC2, are important for neuronal as well as retinal homeostasis, respectively. TOPORS is ubiquitously expressed, yet its mutations are only known to result in autosomal dominant retinitis pigmentosa. We performed a yeast two-hybrid (Y2H) screen of a human retinal cDNA library in order to identify interacting protein partners of TOPORS from the retina, and thus begin delineating the putative disease mechanism(s) associated with the retina-specific phenotype resulting from mutations in TOPORS. The screen led to isolation of the 26 S protease regulatory subunit 4 (P26s4/ PSMC1), an ATPase indispensable for correct functioning of UPS-mediated proteostasis. The interaction between endogenous TOPORS and P26s4 proteins was validated by co-immuno-precipitation from mammalian cell extracts and further characterised by immunofluorescent co-localisation studies in cell lines and retinal sections. Findings from hTERT-RPE1 and 661W cells demonstrated that TOPORS and P26s4 co-localise at the centrosome in cultured cells. Immunofluorescent staining of mouse retinae revealed a strong P26s4 reactivity at the interface between retinal pigmented epithelium (RPE) layer and the photoreceptors outer segments (OS). This finding leads us to speculate that P26s4, along with TOPORS, may have a role(s) in RPE phagocytosis, in addition to contributing to the overall photoreceptor and retinal homeostasis via the UPS.

Study Molecular Interactions in whole Cell Extracts by Fluorescence-Detected Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a classical biophysical technique to characterize proteins and their interactions in solution. Traditional absorbance and interference detection systems lack specificity and high sensitivity, which limit AUC applications in complex and concentrated solutions. A recently introduced fluorescence detection system (FDS) strongly increases the sensitivity of the detection in AUC, and offers the potential to study proteins and protein interactions in biological fluids. By using enhanced green fluorescent protein (EGFP) as a model system, we explore the potential and challenges posed by the application of FDS-AUC to the study of protein interactions in E.coli and Cos7 whole cell extracts. Complex autofluorescence and quenching processes limit the concentration of E.coli cell extract to a few mg/mL. At these concentrations of E.coli cell extract, no impact of this environment on the sedimentation of EGFP is discernable. Autofluorescence also sets the detection limit of EGFP in the low nanomolar range. In comparison with E.coli, the mammalian Cos7 cell extract has less background autofluorescence, which lowers the detection limit for EGFP into the subnanomolar range. To investigate protein interactions, monoclonal anti-EGFP antibody was added to the E.coli cell extract with exogenous EGFP and the Cos7 cell extract with exogenous or endogenous EGFP. All three arrangements show shifts in the sedimentation coefficient distribution that reflect complex formation. Quantitatively, a small decrease of the complex sedimentation coefficient can be observed in cell extracts due to hydrodynamic nonideality, still allowing the determination of complex size and stoichiometry. In summary, this study demonstrates the potential for studying interactions of unpurified GFP-tagged proteins in bacterial and mammalian cell extracts using fluorescence detected AUC.

Well-defined nanostructured surface-imprinted polymers for the highly selective enrichment of low-abundance protein in mammalian cell extract

A new approach for the adsorption and enrichment of natural low-abundance protein by using nanostructured surface-imprinted polymers is presented.

Loading of Argonaute Protein with Small Duplex RNA in Cellular Extracts.

Argonaute (Ago) proteins are the minimum core proteins required for executing RNA interference (RNAi) mechanisms of gene regulation. For Ago proteins to regulate gene expression through RNAi they must be loaded, or "programmed," with a single strand of small RNA. Natural small RNAs are typically double-stranded duplexes that require additional factors for efficient and specific loading into Ago proteins. Here, a protocol is described for investigating RNAi programming through loading of human Ago2 using radiolabeled small interfering RNA (siRNA) and HeLa cell extracts. This protocol provides an Ago loading assay to study RNAi programming when starting with crude or partially purified cell extracts. The Ago loading assay should prove useful for studying other Ago proteins using a variety of mammalian cell extracts.

Erratum: Corrigendum: Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts

Erratum: Corrigendum: Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts

TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

The microtubule-associated protein, TPX2, regulates the activity of the mitotic kinesin, Eg5, but the mechanism of regulation is not established. Using total internal reflection fluorescence microscopy, we observed that Eg5, in extracts of mammalian cells expressing Eg5-EGFP, moved processively toward the microtubule plus-end at an average velocity of 14 nm/s. TPX2 bound to microtubules with an apparent dissociation constant of ∼ 200 nm, and microtubule binding was not dependent on the C-terminal tails of tubulin. Using single molecule assays, we found that full-length TPX2 dramatically reduced Eg5 velocity, whereas truncated TPX2, which lacks the domain that is required for the interaction with Eg5, was a less effective inhibitor at the same concentration. To determine the region(s) of Eg5 that is required for interaction with TPX2, we performed microtubule gliding assays. Dimeric, but not monomeric, Eg5 was differentially inhibited by full-length and truncated TPX2, demonstrating that dimerization or residues in the neck region are important for the interaction of TPX2 with Eg5. These results show that both microtubule binding and interaction with Eg5 contribute to motor inhibition by TPX2 and demonstrate the utility of mammalian cell extracts for biophysical assays.

Mammalian CAP interacts with CAP, CAP2, and actin.

We previously identified human CAP, a homolog of the yeast adenylyl cyclase-associated protein. Previous studies suggest that the N-terminal and C-terminal domains of CAP have distinct functions. We have explored the interactions of human CAP with various proteins. First, by performing yeast two-hybrid screens, we have identified peptides from several proteins that interact with the C-terminal and/or the N-terminal domains of human CAP. These peptides include regions derived from CAP and BAT3, a protein with unknown function. We have further shown that MBP fusions with these peptides can associate in vitro with the N-terminal or C-terminal domains of CAP fused to GST. Our observations indicate that CAP contains regions in both the N-terminal and C-terminal domains that are capable of interacting with each other or with themselves. Furthermore, we found that myc-epitope-tagged CAP coimmunoprecipitates with HA-epitope-tagged CAP from either yeast or mammalian cell extracts. Similar results demonstrate that human CAP can also interact with human CAP2. We also show that human CAP interacts with actin, both by the yeast two-hybrid test and by coimmunoprecipitation of epitope-tagged CAP from yeast or mammalian cell extracts. This interaction requires the C-terminal domain of CAP, but not the N-terminal domain. Thus CAP appears to be capable of interacting in vivo with other CAP molecules, CAP2, and actin. We also show that actin co-immunoprecipitates with HA-CAP2 from mammalian cell extracts.

A peptide affinity reagent for isolating an intact and catalytically active multi-protein complex from mammalian cells

We have developed an approach for directly isolating an intact multi-protein chromatin remodeling complex from mammalian cell extracts using synthetic peptide affinity reagent 4. FOG1(1–15), a short peptide sequence known to target subunits of the nucleosome remodeling and deacetylase (NuRD) complex, was joined via a 35-atom hydrophilic linker to the StreptagII peptide. Loading this peptide onto Streptactin beads enabled capture of the intact NuRD complex from MEL cell nuclear extract. Gentle biotin elution yielded the desired intact complex free of significant contaminants and in a form that was catalytically competent in a nucleosome remodeling assay. The efficiency of 4 in isolating the NuRD complex was comparable to other reported methods utilising recombinantly produced GST–FOG1(1–45).

In vitro replication assay with mammalian cell extracts.

Regulatory mechanisms are crucial to control DNA replication during cell cycle in eukaryotic cells. Cell-free in vitro replication assay (IVRA) is one of the widely used assays to understand the complex mammalian replication system. IVRA can provide a snapshot of the regulatory mechanisms controlling replication in higher eukaryotes by using a single plasmid, pEPI-1. This chapter outlines the general strategies and protocols used to perform IVRA to study the differential recruitment of replication factors either independently or in combination, based on the experience in studying the role of prohibitin in replication as well as other published protocols. This method can be employed to identify not only proteins that assist replication but also proteins that inhibit replication of mammalian genome.

Post-Translational Modification Profiling--a High-Content Assay for Identifying Protein Modifications in Mammalian Cellular Systems.

Protein microarrays are extremely useful for detecting substrates of phosphorylation, substrates of ubiquitylation, or other post-translational modifications. The ability to screen binding interactions as well as post-translational modifications of thousands of proteins at once has improved our ability to identify their targets. Utilizing such systems in combination with functional mammalian cell extracts that preserve enzymatic activity offers advantages in identifying semi-quantitative changes of these interactions in the context of specific cellular conditions. This unit provides a detailed procedure for setting up an extract-based activity assay for high content detection of protein post-translation modifications. It also provides basic guidelines for data analysis.