CRISPR Cas13-based tools to track and manipulate endogenous telomeric repeat-containing RNAs in living cells

TERRA, TElomeric Repeat-containing RNA, is a long non-coding RNA transcribed from telomeres. Emerging evidence indicates that TERRA regulates telomere maintenance and chromosome end protection in normal and cancerous cells. However, the mechanism of how TERRA contributes to telomere functions is still unclear, partially owing to the shortage of approaches to track and manipulate endogenous TERRA molecules in live cells. Here, we developed a method to visualize TERRA in live cells via a combination of CRISPR Cas13 RNA labeling and Suntag technology. Single-particle tracking reveals that TERRA foci undergo anomalous diffusion in a manner that depends on the timescale and telomeric localization. Furthermore, we used a chemically-induced protein dimerization system to manipulate TERRA subcellular localization in live cells. Overall, our approaches to monitor and control TERRA locations in live cells provide powerful tools to better understand its roles in telomere maintenance and genomic integrity.

Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.

mem-iLID, a fast and economic protein purification method

Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system iLID, which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the protein of interest, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.

Evaluating the Efficiency of the Martini Force Field to Study Protein Dimerization in Aqueous and Membrane Environments

<p>Protein-protein complex assembly is one of the major drivers of biological response. Understanding the mechanisms of protein oligomerization/dimerization would allow one to elucidate how these complexes participate in biological activities and could ultimately lead to new approaches in designing novel therapeutic agents. However, determining the exact association pathways and structures of such complexes remains a challenge. Here, we use parallel tempering metadynamics simulations in the well-tempered ensemble to evaluate the performance of Martini 2.2P and Martini open-beta 3 (Martini 3) force fields in reproducing the structure and energetics of the dimerization process of membrane proteins and proteins in an aqueous solution in reasonable accuracy and throughput. We find that Martini 2.2P systematically overestimates the free energy of association by estimating large barriers in distinct areas, which likely leads to overaggregation when multiple monomers are present. In comparison, the less viscous Martini 3 results in a systematic underestimation of the free energy of association for proteins in solution, while it performs well in describing the association of membrane proteins. In all cases the near-native dimer complexes are identified as minima in the free energy surface albeit not always as the lowest minima. In the case of Martini 3 we find that the spurious supramolecular protein aggregation present in Martini 2.2P multimer simulations is alleviated and thus this force field may be more suitable for the study of protein oligomerization. We propose that the use of enhanced sampling simulations with a refined coarse-grained force field and appropriately defined collective variables is a robust approach for studying the protein dimerization process, although one should be cautious of the ranking of energy minima.</p>

Evaluating the Efficiency of the Martini Force Field to Study Protein Dimerization in Aqueous and Membrane Environments

<p>Protein-protein complex assembly is one of the major drivers of biological response. Understanding the mechanisms of protein oligomerization/dimerization would allow one to elucidate how these complexes participate in biological activities and could ultimately lead to new approaches in designing novel therapeutic agents. However, determining the exact association pathways and structures of such complexes remains a challenge. Here, we use parallel tempering metadynamics simulations in the well-tempered ensemble to evaluate the performance of Martini 2.2P and Martini open-beta 3 (Martini 3) force fields in reproducing the structure and energetics of the dimerization process of membrane proteins and proteins in an aqueous solution in reasonable accuracy and throughput. We find that Martini 2.2P systematically overestimates the free energy of association by estimating large barriers in distinct areas, which likely leads to overaggregation when multiple monomers are present. In comparison, the less viscous Martini 3 results in a systematic underestimation of the free energy of association for proteins in solution, while it performs well in describing the association of membrane proteins. In all cases the near-native dimer complexes are identified as minima in the free energy surface albeit not always as the lowest minima. In the case of Martini 3 we find that the spurious supramolecular protein aggregation present in Martini 2.2P multimer simulations is alleviated and thus this force field may be more suitable for the study of protein oligomerization. We propose that the use of enhanced sampling simulations with a refined coarse-grained force field and appropriately defined collective variables is a robust approach for studying the protein dimerization process, although one should be cautious of the ranking of energy minima.</p>

Loss of Cysteine Rich domain was critical for evolution of heterodimerization in Toll proteins

Toll proteins play roles in immunity/development which have largely remained conserved. However, there are differences in Toll biology as mammalian TLRs recognise pathogen associated molecular patterns (PAMPs) but not their invertebrate homologues. The reason for the same is not known. One critical molecular difference is absence of Cysteine Rich Domain (CRD) in vertebrate Tolls and their presence in invertebrates. Interestingly, in Drosophila, all Toll proteins have CRD except Toll9. This provided us the appropriate model to investigate significance of loss of CRD in Toll evolution. CRDs nudge protein dimerization by forming disulphide bonds hence we asked if they did same in Drosophila Toll-proteins. We tested if, Toll-1(which forms homodimer) can heterodimerize with Toll-9. We found that wildtype Toll-1 didn’t interact with Toll9 however; when CRD of Toll1 was deleted/mutated it formed heterodimer with Toll9. This indicates that presence of CRD limits Toll proteins to form homodimer and thus its loss was a critical event which pushed Toll proteins towards heterodimerization. We further show that Drosophila Toll9 can directly bind dsRNA, a PAMP. Interestingly, dsRNA affinity for toll-9 monomer was twice as that for the dimer, which can be attributed to CRD loss. Thus, we show that loss of CRD was a major step in Toll evolution as it resulted in functional diversity and was a first step towards heterodimer formation. Therefore, we propose that CRD loss was under positive selection and also that heterodimerization of Toll-proteins is an evolved property.One line summaryLoss of Cysteine Rich Domain in Drosophila Toll9 and recognition of dsRNA.

W196 and the β-Hairpin Motif Modulate the Redox Switch of Conformation and the Biomolecular Interaction Network of the Apoptosis-Inducing Factor

The human apoptosis-inducing factor (hAIF) is a moonlight flavoprotein involved in mitochondrial respiratory complex assembly and caspase-independent programmed cell death. These functions might be modulated by its redox-linked structural transition that enables hAIF to act as a NAD(H/+) redox sensor. Upon reduction with NADH, hAIF undergoes a conformational reorganization in two specific insertions—the flexible regulatory C-loop and the 190-202 β-harpin—promoting protein dimerization and the stabilization of a long-life charge transfer complex (CTC) that modulates its monomer-dimer equilibrium and its protein interaction network in healthy mitochondria. In this regard, here, we investigated the precise function of the β-hairpin in the AIF conformation landscape related to its redox mechanism, by analyzing the role played by W196, a key residue in the interaction of this motif with the regulatory C-loop. Mutations at W196 decrease the compactness and stability of the oxidized hAIF, indicating that the β-hairpin and C-loop coupling contribute to protein stability. Kinetic studies complemented with computational simulations reveal that W196 and the β-hairpin conformation modulate the low efficiency of hAIF as NADH oxidoreductase, contributing to configure its active site in a noncompetent geometry for hydride transfer and to stabilize the CTC state by enhancing the affinity for NAD+. Finally, the β-hairpin motif contributes to define the conformation of AIF’s interaction surfaces with its physiological partners. These findings improve our understanding on the molecular basis of hAIF’s cellular activities, a crucial aspect for clarifying its associated pathological mechanisms and developing new molecular therapies.