A Step-by-Step Guide for the Production of Recombinant Fluorescent TAT-HA-Tagged Proteins and their Transduction into Mammalian Cells.

Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step-by-step the engineering of a recombinant protein with various tags: TAT-HA (trans-activator of transduction-hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni-nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT-HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step-by-step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein production Basic Protocol 2: Protein purification Basic Protocol 3: Protein transduction in mammalian cells.

Two Tau binding sites on tubulin revealed by thiol-disulfide exchanges.

Tau is a Microtubule-associated protein that induces and stabilizes the formation of the Microtubule cytoskeleton and plays an important role in neurodegenerative diseases. The Microtubules binding region of Tau has been determined for a long time but where and how Tau binds to its partner still remain a topic of debate. We used Site Directed Spin Labeling combined with EPR spectroscopy to monitor Tau upon binding to either Taxol-stabilized MTs or to αß-tubulin when Tau is directly used as an inducer of MTs formation. Using maleimide-functionalized labels grafted on the two natural cysteine residues of Tau, we found in both cases that Tau remains highly flexible in these regions confirming the fuzziness of Tau:MTs complexes. More interestingly, using labels linked by a disulfide bridge, we evidenced for the first time thiol disulfide exchanges between αß-tubulin or MTs and Tau. Additionally, Tau fragments having the two natural cysteines or variants containing only one of them were used to determine the role of each cysteine individually. The difference observed in the label release kinetics between preformed MTs or Tau-induced MTs, associated to a comparison of structural data, led us to propose two putative binding sites of Tau on αß-tubulin.

Modification by SUMOylation Controls Both the Transcriptional Activity and the Stability of Delta-Lactoferrin.

Delta-lactoferrin is a transcription factor, the expression of which is downregulated or silenced in case of breast cancer. It possesses antitumoral activities and when it is re-introduced in mammary epithelial cancer cell lines, provokes antiproliferative effects. It is posttranslationally modified and our earlier investigations showed that the O-GlcNAcylation/phosphorylation interplay plays a major role in the regulation of both its stability and transcriptional activity. Here, we report the covalent modification of delta-lactoferrin with the small ubiquitin-like modifier SUMO-1. Mutational and reporter gene analyses identified five different lysine residues at K13, K308, K361, K379 and K391 as SUMO acceptor sites. The SUMOylation deficient M5S mutant displayed enhanced transactivation capacity on a delta-lactoferrin responsive promoter, suggesting that SUMO-1 negatively regulates the transactivation function of delta-lactoferrin. K13, K308 and K379 are the main SUMO sites and among them, K308, which is located in a SUMOylation consensus motif of the NDSM-like type, is a key SUMO site involved in repression of delta-lactoferrin transcriptional activity. K13 and K379 are both targeted by other posttranslational modifications. We demonstrated that K13 is the main acetylation site and that favoring acetylation at K13 reduced SUMOylation and increased delta-lactoferrin transcriptional activity. K379, which is either ubiquitinated or SUMOylated, is a pivotal site for the control of delta-lactoferrin stability. We showed that SUMOylation competes with ubiquitination and protects delta-lactoferrin from degradation by positively regulating its stability. Collectively, our results indicate that multi-SUMOylation occurs on delta-lactoferrin to repress its transcriptional activity. Reciprocal occupancy of K13 by either SUMO-1 or an acetyl group may contribute to the establishment of finely regulated mechanisms to control delta-lactoferrin transcriptional activity. Moreover, competition between SUMOylation and ubiquitination at K379 coordinately regulates the stability of delta-lactoferrin toward proteolysis. Therefore SUMOylation of delta-lactoferrin is a novel mechanism controlling both its activity and stability.

The FK506-binding protein FKBP52 in vitro induces aggregation of truncated Tau forms with prion-like behavior.

Tauopathies, including Alzheimer's disease (AD), are neurodegenerative diseases associated with the pathologic aggregation of human brain Tau protein. Neuronal Tau is involved in microtubule (MT) formation and stabilization. We showed previously that the immunophilin FK506-binding protein of MW ∼52 kDa (FKBP52) interferes with this function of full-length Tau and provokes aggregation of a disease-related mutant of Tau. To dissect the molecular interaction between recombinant human FKBP52 and Tau, here, we study the effect of FKBP52 on a functional Tau fragment (Tau-F4, Ser(208)-Ser(324)) containing part of the proline- rich region and MT-binding repeats. Therefore, we perform MT assembly and light-scattering assays, blue native PAGE analysis, electron microscopy, and Tau seeding experiments in SH-SY5Y human neuroblastoma cells. We show that FKBP52 (6 µM) prevents MT formation generated by Tau-F4 (5 µM) and induces Tau-F4 oligomerization and aggregation. Electron microscopy analyses show granular oligomers and filaments of Tau-F4 after short-time FKBP52 incubation. We demonstrate that the terminal parts of Tau interfere with the effects of FKBP52. Finally, we find that FKBP52-induced Tau-F4 oligomers cannot only generate in vitro, direct conformational changes in full-length Tau and that their uptake into neuronal cells can equally lead to aggregation of wild-type endogenous Tau. This suggests a potential prion-like property of these particular Tau-F4 aggregates. Collectively, our results strengthen the hypothesis of FKBP52 involvement in the Tau pathogenicity process.

Tau monoclonal antibody generation based on humanized yeast models: impact on Tau oligomerization and diagnostics.

A link between Tau phosphorylation and aggregation has been shown in different models for Alzheimer disease, including yeast. We used human Tau purified from yeast models to generate new monoclonal antibodies, of which three were further characterized. The first antibody, ADx201, binds the Tau proline-rich region independently of the phosphorylation status, whereas the second, ADx215, detects an epitope formed by the Tau N terminus when Tau is not phosphorylated at Tyr(18). For the third antibody, ADx210, the binding site could not be determined because its epitope is probably conformational. All three antibodies stained tangle-like structures in different brain sections of THY-Tau22 transgenic mice and Alzheimer patients, and ADx201 and ADx210 also detected neuritic plaques in the cortex of the patient brains. In hippocampal homogenates from THY-Tau22 mice and cortex homogenates obtained from Alzheimer patients, ADx215 consistently stained specific low order Tau oligomers in diseased brain, which in size correspond to Tau dimers. ADx201 and ADx210 additionally reacted to higher order Tau oligomers and presumed prefibrillar structures in the patient samples. Our data further suggest that formation of the low order Tau oligomers marks an early disease stage that is initiated by Tau phosphorylation at N-terminal sites. Formation of higher order oligomers appears to require additional phosphorylation in the C terminus of Tau. When used to assess Tau levels in human cerebrospinal fluid, the antibodies permitted us to discriminate patients with Alzheimer disease or other dementia like vascular dementia, indicative that these antibodies hold promising diagnostic potential.

SILAC-based proteomic profiling of the human MDA-MB-231 metastatic breast cancer cell line in response to the two antitumoral lactoferrin isoforms: the secreted lactoferrin and the intracellular delta-lactoferrin.

BACKGROUND: Lactoferrins exhibit antitumoral activities either as a secretory lactoferrin or an intracellular delta-lactoferrin isoform. These activities involve processes such as regulation of the cell cycle and apoptosis. While lactoferrin has been shown to exert its function by activating different transduction pathways, delta-lactoferrin has been proven to act as a transcription factor. Like many tumor suppressors, these two proteins are under-expressed in several types of cancer, particularly in breast cancer. METHODOLOGY/PRINCIPAL FINDINGS: In order to compare the differential effects of the re-introduction of lactoferrin isoforms in breast cancer cells we chose the cancerous mammary gland MDA-MB-231 cell line as a model. We produced a cell line stably expressing delta-lactoferrin. We also treated these cells with fresh purified human breast lactoferrin. We performed two quantitative proteomic studies in parallel using SILAC coupled to mass spectrometry in order to compare the effects of different doses of the two lactoferrin isoforms. The proteome of untreated, delta-lactoferrin expressing and human lactoferrin treated MDA-MB-231 cells were compared. Overall, around 5300 proteins were identified and quantified using the in-house developed MFPaQ software. Among these, expression was increased by 1.5-fold or more for around 300 proteins in delta-lactoferrin expressing cells and 190 proteins in lactoferrin treated cells. At the same time, about 200 and 40 proteins were found to be downregulated (0-0.7-fold) in response to delta-lactoferrin and lactoferrin, respectively. CONCLUSIONS/SIGNIFICANCE: Re-introduction of delta-lactoferrin and lactoferrin expression in MDA-MB-231 mainly leads to modifications of protein profiles involved in processes such as proliferation, apoptosis, oxidative stress, the ubiquitin pathway, translation and mRNA quality control. Moreover, this study identified new target genes of delta-lactoferrin transcriptional activity such as SelH, GTF2F2 and UBE2E1.

Nuclear magnetic resonance analysis of the acetylation pattern of the neuronal Tau protein.

Lysine acetylation of the neuronal Tau protein was described as a novel mechanism of posttranslational regulation of Tau functions with important outcomes in microtubule binding and aggregation processes related to Alzheimer's disease. Here, we unravel at a per-residue resolution the acetylation pattern of full-length Tau by the Creb-binding protein (CBP) acetyltransferase using high-resolution nuclear magnetic resonance spectroscopy. Our study gives a quantitative overview of CBP-mediated acetylation and examines the catalytic proficiency because the nonenzymatic reaction with acetyl-coenzyme A occurs in vitro. Furthermore, we have investigated with this characterized acetylated Tau the effect of acetylation on Tau fibrillization in a heparin-induced aggregation assay and on heparin binding.

A functional fragment of Tau forms fibers without the need for an intermolecular cysteine bridge.

We study the aggregation of a fragment of the neuronal protein Tau that contains part of the proline rich domain and of the microtubule binding repeats. When incubated at 37 °C with heparin, the fragment readily forms fibers as witnessed by Thioflavin T fluorescence. Electron microscopy and NMR spectroscopy show bundled ribbon like structures with most residues rigidly incorporated in the fibril. Without its cysteines, this fragment still forms fibers of a similar morphology, but with lesser Thioflavin T binding sites and more mobility for the C-terminal residues.

Delta-lactoferrin, an intracellular lactoferrin isoform that acts as a transcription factor.

Delta-lactoferrin (ΔLf) is a transcription factor of which the expression is downregulated in cancer. It is a healthy tissue marker and a high expression level of its transcripts was correlated with a good prognosis in breast cancer. ΔLf results from alternative promoter usage of the hLf gene leading to the production of 2 isoforms with alternative N-termini: lactoferrin, which is secreted, and ΔLf, its nucleocytoplasmic counterpart. ΔLf possesses antiproliferative properties and induces cell cycle arrest. It is an efficient transcription factor interacting in vivo via a ΔLf response element found in the Skp1, Bax, DcpS, and SelH promoters. Since ΔLf possesses different target genes, modifications in its activity or concentration may have crucial effects on cell homeostasis. Posttranslational modifications modulate ΔLf transcription factor activity. Our earlier investigations showed that O-GlcNAcylation negatively regulates ΔLf transcriptional activity, whilst inhibiting its ubiquitination and increasing its half-life. On the other hand, phosphorylation potentiates ΔLf transcriptional activity. Recently, we showed that ΔLf is also modified by SUMOylation. Therefore, cooperation and (or) competition among SUMOylation, ubiquitination, phosphorylation, and O-GlcNAcylation may contribute to the establishment of a fine regulation of ΔLf transcriptional activity depending on the type of target gene and cellular homeostasis.

Expression, purification, and structural prediction of the Ets transcription factor ERM.

The PEA3 group within the Ets family comprises PEA3, ER81, and ERM, three transcription factors of about 500 residues. These factors are highly conserved in their ETS DNA-binding domain and in their two transcriptional activation domains. They are involved in many developmental processes and regulate cancer development via metastasis, as in the case of some breast tumors. Here, we describe the oversynthesis of human ERM from a baculovirus expression vector in Spodoptera frugiperda (Sf9) cells, and the subsequent purification and structural characterization of this protein. Oversynthesis of ERM was confirmed by measuring band intensities on SDS-PAGE gels and by Western blot analysis. Two-step purification by affinity chromatography led to a highly stable protein. Electromobility shift assays suggested that this purified protein is functional, since it recognizes specific Ets DNA-binding sites. We then used circular dichroism and infrared spectrometry to perform a structural analysis of the purified full-length ERM, and compared the results with those of current structural prediction algorithms. Our study indicates that ERM contains a highly structured ETS-domain and suggests that each of the N- and C-terminal transactivating domains also contains an alpha-helix. In contrast, the 250-residue central domain seems to have very little structure.