Kanazawa University NanoLSI Podcast Adarsh Sandhu

Researchers observe what ubiquitination hinges on 
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Hiroki Konno and Holger Flechsig at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University.
The research described in this podcast was published in Nano Letters in December 2023
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Researchers observe what ubiquitination hinges on
Researchers at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University report in Nano Letters how the flexibility of a protein hinge plays a crucial role in the transfer of proteins in key cell processes.
Ubiquitination – the addition of the protein ubiquitin – is a key stage in many cell processes, such as protein degradation, DNA repairs, and signal transduction. Using high-speed atomic force microscopy (AFM) and molecular modelling, researchers led by Hiroki Konno and Holger Flechsig at WPI-NanoLSI, Kanazawa University have identified how the mobility of a ubiquitination related enzyme hinge allows ubiquitination to take place.
So what was known already about ubiquitination?
Previous studies have identified a number of enzymes that facilitate ubiquitination, including an enzyme that activates ubiquitin (E1), an enzyme that conjugates it (E2), and an enzyme that catalyzes ubiquitin protein joining (that is, a ligase, E3) to a target protein. The HECT-type E3 ligase is characterized by a HECT domain that comprises an N lobe with the E2-binding site and a C lobe with a catalytic Cys residue, A flexible hinge connects the two lobes, leading to the hypothesis that ubiquitination is facilitated by the rearrangement of the protein around this hinge. Konno and their collaborators deployed their high-speed atomic force microscope to hunt for evidence that this was the case.
So what did they find out?
The researchers noted that when the HECT domain was crystallized with a type of E2 enzyme, it formed an L shape such that the distance between the catalytic Cys residue of the HECT domain and the catalytic Cys of the E2 enzyme was 41 Å – too far for the transfer of ubiquitin. However, in its catalytic conformation the HECT domain has a different shape where the distance between the two catalytic Cys residues is much closer – just 8 Å – so this is thought to be a “catalytic conformation”.
Analysis of high-speed-AFM images of a wild-type HECT domain of E6AP revealed two conformations – one of which looked spherical and the other oval. Using AFM simulations they attributed the oval shapes to the L conformation and spherical shapes are either the catalytic conformation or the so called inverted T conformation, which had been observed in the another type of HECT domain where the distance between the Cys residues is 16 Å. To overcome the spatio-temporal resolution limitations of imaging, the experiments were complemented by molecular modelling to visualize HECT domain conformational motions at the atomistic level. Simulation AFM was used to generate a corresponding pseudo AFM movie, which clearly showed the change from spherical to the oval shaped topography.
“Although experimental limitations do not allow us to resolve the intermediate conformations,” explain the researchers in their report of the work. “The performed modeling provides evidence that the transitions between spherical and oval HECT domain shapes observed under high-speed-AFM correspond to functional conformational motions under which the C-lobe rotates relative to the N-lobe, thereby allowing the change between catalytic and L-shape HECT conformations.”
Further experiments with mutant HECT domains with less flexibility in the h
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Chromatin Accessibility: A new avenue for gene editing
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by researchers from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, led by Yusuke Miyanari.
The research described in this podcast was published in Nature Genetics in February 2024
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https://nanolsi.kanazawa-u.ac.jp/en/
Chromatin Accessibility: A new avenue for gene editing
In a study recently published in Nature Genetics, researchers from Nano Life Science Institute (WPI-NanoLSI) at Kanazawa University explore chromatin accessibility, that is, endogenous access pathways to the genomic DNA, and its use as a tool for gene editing.
Our DNA is protected from unwanted external modifications by forming structures called nucleosomes that consist of threads of DNA wound around chunks of special proteins known as histones. This unique coiled shape prevents the access of undesirable molecules to a cell’s DNA. However, for vital genetic functions—such as DNA repair—the right set of proteins require access to these DNA fragments. This phenomenon known as ‘chromatin accessibility’ involves a privileged set of protein molecules, many of which are still unknown.
Now, researchers from Nano Life Science Institute (WPI-NanoLSI) at Kanazawa University, led by Yusuke Miyanari, have used advanced genetic screening methods to unravel chromatin accessibility and its pathways.
So how did they go about it?
For the investigation the team used a combination of two technologies—CRISPR screening and ATAC-see. While the former is a method to suppress the function of a desired set of genes, the latter is a means to identify which ones are essential for chromatin accessibility. Thus, using this method all genes playing a crucial role in chromatin accessibility could be pinned down.
With the help of these assays, novel pathways and individual players involved in chromatin accessibility were uncovered—some playing a positive role and some negative. Of these, one particular protein, TFDP1, showed a negative effect on chromatin accessibility. When it was suppressed, a significant increase in chromatin accessibility was observed, accompanied by nucleosome reduction. A deeper dive into the mechanism of TFDP1 revealed that it functions by regulating the genes responsible for production of certain histone proteins.
The team then focused their study towards exploring biotechnological applications of their findings. After suppressing TFDP1, two different approaches were tried. The first approach involved gene editing using the CRISPR/Cas9 tool. This revealed that deletion of TFDP1 made the gene editing process easier. Now, most chromatin accessibility occurs in nucleosome-depleted regions or NDRs. However, by suppressing TFDP1 chromatin accessibility occurred not only in NDRs but across other regions as well. Secondly, the depletion of TFDP1 aided the process of converting regular cells into stem cells, a massive step forward in cellular transformation.
This study identified new chromatin accessibility pathways and channels for exploring its potential even further. “Our study shows the significant potential to manipulate chromatin accessibility as a novel strategy to enhance DNA-templated biological applications, including genome editing and cellular reprogramming,” conclude the researchers.
Reference
Satoko Ishii, Taishi Kakizuka, Sung-Joon Park, Ayako Tagawa, Chiaki Sanbo, Hideyuki Tanabe, Yasuyuki Ohkawa, Mahito Nakanishi, Kenta Nakai, Yusuke Miyanari. Genome-wide ATAC-see screening identifies TFDP1 as a modulator of global chromatin accessibility. Nature Genetics, Feb
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Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Romain Amyot, Noriyuki Kodera, and Holger Flechsig at the Kanazawa University NanoLSI.
The research described in this podcast was published in Frontiers in Molecular Biosciences  in November 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Researchers predict protein placement on AFM substrates
Researchers at Kanazawa University report in Frontiers in Molecular Biosciences a computational method to predict the placement of proteins on AFM substrates based on electrostatic interactions.
The observation of biomolecular structures using atomic force microscopy (AFM) and the direct visualization of functional conformational dynamics in high-speed AFM (HS-AFM) experiments have significantly advanced the understanding of biological processes at the nanoscale. In experiments, a biological sample is deposited on a supporting surface (AFM substrate) and is scanned by a probing tip to detect the molecular shape and its dynamical changes. The observation of protein dynamics under HS-AFM is a delicate balance between immobilizing the structure on the supporting surface while at the same time preventing too strong perturbations by immobilization.
The process of placing a biomolecular sample on the supporting surface and controlling its proper attachment is a challenge at the very start of every AFM observation. By the chemical composition of the buffer, interactions between the sample and substrate can be modified. Such surface modifications are often critical for successful AFM observations of protein structures and their functional motions. However, the molecular orientation of the sample is a priori unknown, and due to limitations in the spatial resolution of images, difficult to infer from a posteriori analysis.
Romain Amyot, Noriyuki Kodera, and Holger Flechsig from Kanazawa University have now developed a physical model to predict the placement of biomolecular structures on AFM substrates based on electrostatic interactions. The method considers the substrates commonly used in AFM experiments (mica, APTES-mica, lipid bilayers) and takes into account buffer conditions. In computer simulations, a large number of possible molecular arrangements on the AFM substrate are sampled, and from evaluating the corresponding interaction energies, the most favorable placement is determined. Furthermore, the analysis allows predictions of the imaging stability under tip scanning.
The authors provide several applications of the new method and obtain remarkable agreement of model predictions with previous experimental HS-AFM imaging of proteins. The findings can explain, for example, why HS-AFM observations of the Cas9 endonuclease, a protein playing a key role in genetic engineering applications, can reliably visualize functional relative motions of target DNA and Cas9 and capture DNA cleavage events at the single molecule level (see Fig. 1). Furthermore, as demonstrated for the ATP-powered chaperone machine ClpB, the model can explain how buffer conditions affect the stability of the sample-substrate complex and validate observations of previous HS-AFM experiments.
In summary, the new method allows to employ the enormous amount of available structural data for biomolecules to make predictions of the sample placement on AFM substrates even prior to an actual experiment, and it can also be applied for post-experimental analysis of AFM imaging data. The developed method is implemented within the freely available BioAFMviewer software package, providing a convenient platform for applications by the broad BioAFM community.  
Reference 
R. Amyot, K. Nakamoto, N. Kodera, H. Flechsi
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Sodium channel investigation
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Ayumi Sumino and Takashi Sumikama at the Kanazawa University NanoLSI.
The research described in this podcast was published in Nature Communications in December 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Sodium channel investigation
Researchers at Kanazawa University report in Nature Communications a high-speed atomic force microscopy study of the structural dynamics of sodium ion channels in cell membranes.  The findings provide insights into the mechanism behind the generation of cell-membrane action potentials.
The transport of ions to and from a cell is controlled by pore-forming proteins embedded in the cell membrane.  In particular, so-called voltage-gated sodium channels (VGSCs) govern the transfer of sodium (Na+) ions, and play an important role in the regulation of the membrane potential — the voltage difference between the cell’s exterior and interior.  In electrically excitable cells such as neurons and muscle cells, VGSCs participate in the generation of action potentials; these are rapid changes in the membrane potential enabling the transmission of e.g. neural signals.  The precise structural changes occurring in VGSCs are not completely understood, however.  Now, Ayumi Sumino and Takashi Sumikama from Kanazawa University in collaboration with Katsumasa Irie from Wakayama Medical University and colleagues have succeeded in observing the structural dynamics of VGSC by means of high-speed atomic force microscopy (high speed-AFM), a method capable of imaging the nanostructure and subsecond dynamics of biomolecules.
VGSCs can be in three different states: resting, inactive and active.  In the latter state, Na+ ions can pass through the channel; in the resting and inactive states, which are structurally different, ions cannot pass.  The basic structure of a VGSC consists of two modules: voltage sensor domains and pore domains.  These domains form a square arrangement, with the ion pore at its center.  An important open question is whether the voltage sensor domains dissociate from the pore domains when the channel closes.
So how did they go about determining this?
Sumino and colleagues performed experiments on three VGSCs.  One is the sodium channel of a particular bacterium (Arcobacter butzleri), the other two are mutants of it.  These three VGSCs have different voltage dependencies, with activation voltages starting at -120 mV, -50 mV and 0 mV, so that at the experimental conditions (0 mV), the VGSCs are in different states.
In order to provide insights into the structural dynamics of these three VGSCs, the researchers applied high speed-AFM, a powerful technique for producing image sequences of biochemical compounds.  A single AFM image is generated by laterally moving a tip just above the sample’s surface; during this xy-scanning motion, the tip’s position in the direction perpendicular to the xy-plane (the z-coordinate) will follow the sample’s height profile.  The variation of the z-coordinate of the tip then produces a height map — the image of the sample.  The generation of such AFM images in rapid succession then produces a video recording of the sample.
The HS-AFM results revealed that for the mutant VGSC in the resting state, the voltage sensor domains are indeed dissociated from the pore domains.  Furthermore, the researchers found that the dissociated voltage sensor domains of neighboring channels connect to form pairs — this is referred to as dimerization.
The observation of the dissociation of voltage sensor domains, as well as the dimerization between pore channels,
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Researchers fix the chirality of helical proteins
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Naoki Ousaka, Mark J. MacLachlan and Shigehisa Akine at the Kanazawa University NanoLSI.
The research described in this podcast was published in Nature Communications in October 2023
 Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Researchers fix the chirality of helical proteins
Researchers at Kanazawa University report in Nature Communications how they can control chirality inversion in α helical peptides.
The function of a protein is determined by its structure – prompting great interest in how to manipulate these structures. The structure is defined not just by the sequence of amino acids that make it, but the shape these acids make – the secondary structure – as well as how that shape is then folded. The most common secondary protein structure is the α-helix, which can coil to the right or left. This coiling direction in turn determines how it engages with other chiral structures, which may be the form of a light beam or another molecule. Although molecular components and environmental factors can favor a particular coiling direction over the other, helical molecules tend to flip between the two coil directions. Now Naoki Ousaka, Mark J. MacLachlan and Shigehisa Akine at Kanazawa University in Japan have shown how they can control and fix the coil direction.
Helical proteins are chiral molecules, which means that the molecule’s shape cannot be fitted into its mirror image. In nature helical proteins often have other chiral components, such as sugars or amino acids, and these will determine which way the protein coils. However, there is a lot of interest in synthesizing artificial helical proteins that have different chemical components and hence functions not found in nature, and these may not have other chiral components. Nonetheless having both types or “enantiomers” of the chiral molecule can be hazardous because of the significant differences in behavior between the two chiral forms, one of which may be benign or even therapeutic while the other is toxic. Hence, there is demand for other ways of selecting and fixing the chirality.
So how did they go about this?
Ousaka, MacLachlan and Akine synthesized α helical molecules solely from achiral components. They included bulky segments so that the molecule tended towards the larger rings of the α helical structure, as well as side chains of piperidine – molecular components that are common in pharmaceuticals. These side chains can be cross linked to “staple” the molecule into either the righthanded or lefthanded coil, inhibiting flipping between the two – chiral inversion. Finally they added another molecular component, known as an ester  – the L-Val-OH residue. This would switch the direction of the coil in response to acidic or basic environments due to preferences in the interaction between oxygen atoms in the ester and the amino acid backbone.
The researchers used a range of chiral characterization methods including circular dichroism, nuclear magnetic resonance and liquid chromatography. They found that with the molecule stapled just once, it would slow down the flipping between enantiomers by a factor of 106, although this still occurred over minutes. Changing the solution to acid or alkali also successfully determined which enantiomer was favoured. However, stapling the molecule twice slowed down the chirality inversion by a factor of 1012, so that the molecular chirality was stable for years. This increased energy barrier to chirality inversion could then be overcome by heating the sample to very high temperatures to switch bet
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Genetic switches in tumor development
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Masanobu Oshima at the Kanazawa University NanoLSI.
The research described in this podcast was published in Cancer Research in November 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Genetic switches in tumor development
Researchers at Kanazawa University report in Cancer Research how Kras and p53 mutations influence the tumor suppressor and promoter functions of a TGF- ß pathway. The findings may lead to a new approach for colorectal cancer therapy.
Both the progression and the suppression of tumors are governed by biomolecular processes. Often, a particular process is involved in either cancer progression or suppression. Cancer treatment in the form of drugs then typically focuses on the respective deactivation or activation of the relevant biomolecular process. However, it has been established that a process known as transforming growth factor ß (TGF-ß) signaling*1 plays a role in both tumor suppression and progression. Now, Masanobu Oshima from Kanazawa University and colleagues have studied the precise genetic conditions underlying the outcome of TGF-ß signaling. Their findings may help the development of new therapeutic strategies for particular cancers.
The suppressive effect of TGF-ß signaling happens through the stimulation of cell differentiation — the process through which dividing cells acquire their type or function. The malignant progression of cancers, on the other hand, comes from a process called epithelial-mesenchymal transition (EMT), in which an epithelial cell transforms into a mesenchymal cell type. The former is a ‘stationary’ type of cell, found in epithelial tissue, whereas the latter is a more ‘migratory’ type of cell found in development and cancer.
So how did they investigate these processes and what did they found out?
Oshima and colleagues performed experiments with tumor-derived organoids. They confirmed that TGF-ß family cytokine, activin plays a role in tumor suppression and progression dependent on the mutation types of driver genes. In certain cancer cells treated with activin, the researchers noted that the partial EMT is induced with tumor aggressiveness and development. On the other hand, certain mutated activin receptors were found to have cancer suppressor capabilities, which made the scientists conclude that genetic alterations underlie the dual function of activins.
One of the two relevant genes is Kras which relays signals that regulate cell growth, division and differentiation. Oshima and colleagues found that a mutation of Kras blocks TGF-ß/activin-induced growth suppression. The other gene is known as Trp53, which encodes tumor protein 53, playing an important role in cancer regulation. A combination of Kras and Trp53 mutations at hot spots, known as gain-of-function mutation, was found to not just block tumor suppression but promote partial EMT and tumor proliferation.
The experiments were done with mouse intestinal tumor-derived organoids with defined genetic backgrounds, which makes the results relevant for therapeutic strategies for human colorectal cancer. Quoting the scientists: “Based on these results, the control of TGF- ß/activin signaling appears to be an important preventive and therapeutic strategy against the malignant progression of colorectal cancer carrying […] mutations”.
Reference
Dong Wang, Mizuho Nakayama, Chang Pyo Hong, Hiroko Oshima, and Masanobu Oshima. Gain-of-function p53 mutation acts as a genetic switch for T
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