KR102151145B1 - A method of screening autophagy regulating material using Nd1-L - Google Patents

Abstract

The present invention provides a method for screening a substance that modulates autophagy, comprising the steps of: (a) treating a substance to be tested on nerve cells; And (b) the Nd1-L protein present in the nucleus and cytoplasm is located as a stress aggregate, or the binding of the Nd1-L protein and GABARAP or the binding of the Nd1-L protein and GABARAPL1 is increased compared to the control without treatment with the test substance. It provides a screening method comprising the step of selecting a material to be used.
The screening method for autophagy regulating substances of the present invention identifies the relationship between the stress aggregate of the Nd1-L protein and the activation of autophagy, and is a candidate for the treatment of brain diseases related to the mechanism of regulating stress aggregates in the field of degenerative brain diseases. It can be usefully used when developing materials.

Description

A method of screening autophagy regulating material using Nd1-L}

The present invention relates to a method for screening a substance that regulates autodigestion, and more particularly, through the binding of Nd1-L protein to GABARAP and GABARAPL1 and activation of autodigestion related to stress aggregates of Nd1-L protein thereby. It relates to a method for screening a substance that modulates autodigestion for the prevention or treatment of degenerative nervous system diseases.

The growth of nerve cells is known to be a very active process in which the expansion and contraction of nerve cell axons and dendrites are repeated for proper connection between nerve cells, and it is thought that the regulating action through protein degradation is important in this process.

Autophagy is a pathway that degrades cytoplasmic components such as RNA, protein, or organelles on a large scale in lysosomes (Lee, 2012, Exp Neurobiol 21(1): 1-8; Nixon, 2013, Nat Med 19(8): 983-997), also related to the quality control of proteins and organelles.

The autodigestive pathway is known to be associated with degenerative neurological disorders involving several cytoplasmic inclusions, and in some degenerative neurological disorders, it is known that the accumulation of aggregates and damaged organelles leading to dysfunction of cellular homeostasis and signaling in neurons. have. In addition, oxidative stress is closely related to the onset of ALS, and is known to be important in disease progression (Barber & Shaw, 2010, Free Radic Biol Med 48(5): 629-641).

The Nd1-L protein was first identified as a protein involved in the stabilization of actin filaments (Kazushi Sasagawa et al. 2002, JBC 277(46): 44140-44146), and the expression of the Nd1-L gene was lowered after transcription by the anticancer drug doxorubicin. (Y Takamori et al., 2006, Int J Mol Med 18: 963-967), as a partner that interacts with KRIT1, a major factor in cerebrovascular disease, cerebral cavernous malformations. It plays a role and is known to increase the expression of the antioxidant enzyme SOD2 (Paolo Guazzi et al., 2012, PLOSone 7(9): e44705). In addition, it is known that Nd1-L mRNA coexists as intracellular FUS aggregates in neurons expressing the FUS mutant complex (FUS-R52C complex) associated with ALS, and Nd1-L mRNA is reported to be involved in the recovery of abnormal aggregates. Yes (Jun et al., Jan 2017, SCIENTIFIC REPORTS 7: 40474).

However, until now, little is known about the role of the Nd1-L protein in autophagy and the relationship between the Nd1-L protein and the core protein (LC3/GABARAP), which is an autophagy factor.

Korean Patent Application Publication No. 10-2018-0026154 (2018.03.12.)

Jun et al. SCIENTIFIC REPORTS 2017 Jan, 7: 40474 Y Takamori et al. INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 2006, 18: 963-967 Paolo Guazzi et al. PLOSone 2012, 7(9): e44705 Kazushi Sasagawa et al. JBC 2002, 277(46): 44140-44146

The inventors of the present invention were studying the mechanisms associated with neurodegenerative diseases such as Lou Gehrig's disease or frontotemporal dementia caused by the Nd1-L protein in neurons, while the Nd1-L protein present in the cytoplasm or nucleus in the cell was a stress aggregate. It was found that it was possible to screen for treatments for neurodegenerative diseases such as Lou Gehrig's disease or frontotemporal dementia through a mechanism that activates autodigestion by binding to GABARAP or GABARAPL1, which is an autodigestion-related factor.

Accordingly, an object of the present invention is to provide a method for screening a substance regulating autodigestion for the prevention or treatment of degenerative nervous system diseases.

According to an aspect of the present invention, a method for screening an autophagy modulating substance for the prevention or treatment of degenerative nervous system diseases, comprising: (a) treating a substance to be tested on nerve cells; And (b) the Nd1-L protein present in the nucleus and cytoplasm is located as a stress aggregate, or the binding of the Nd1-L protein and GABARAP or the binding of the Nd1-L protein and GABARAPL1 is increased compared to the control group not treated with the test substance. There is provided a screening method comprising the step of selecting a material to be used.

In one embodiment, the position of the Nd1-L protein in step (b) may be measured by immunocytochemistry.

In one embodiment, the material selected in step (b) may activate autodigestion to control abnormal accumulation of stress aggregates.

In one embodiment, the degenerative nervous system disease may be one selected from the group consisting of Lou Gehrig's disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, and frontotemporal dementia.

According to the present invention, the Nd1-L protein is positioned as a stress aggregate by inducing stress, or by increasing the binding of the Nd1-L protein and GABARAP or the binding of the Nd1-L protein and GABARAPL1, the selective autodigestion-related factors NBR1, p62 And by reducing or increasing the expression of GABARAPL1 to regulate autodigestion, it has been found that abnormal accumulation of stress aggregates in degenerative brain diseases can be regulated.

Therefore, the method of screening for autophagy regulating substances of the present invention identifies the relationship between the stress aggregate of Nd1-L protein and the activation of autophagy, thereby treating brain diseases related to the stress aggregate regulatory mechanism in the field of degenerative brain diseases. It can be usefully used when developing candidate materials for

1 is a neuron in which stress factors (sodium arsenite (SA) and dithiothreitol (DTT)) are treated to cause stress, whereby Nd1-L is located as a G3BP-GFP positive stress aggregate. This is an image confirming that.
2 is an image showing that Nd1-L is accumulated in abnormal stress aggregates associated with R521C, a mutant protein associated with ALS and is associated with degenerative brain disease.
3 is a schematic diagram showing that in the protein domain structure of Nd1-L, there is a BTB/POZ domain in the N-terminal domain, and an LIR (LC3-interacting region) motif sequence in the Kelch motif exists.
Figure 4 is a fluorescent protein attached to GFP to LC3 and GABARAP family proteins not located as autophagy to confirm the intracellular binding of Nd1-L and LC3, GABARAP protein in cultured neurons and LIR and 2xLIR of Nd1-L An image result of confirming fluorescence in the nucleus or cytoplasm by expressing the RFP protein with 3xNLS inserted in the cell (A) and a graph showing a quantitative analysis using the GFP fluorescence intensity for the ratio of the migration of LC3 and GABARAP proteins from the cytoplasm to the nucleus (B) to be.
Figure 5 shows the GST-binding assay results (A) confirming the binding of GST-LC3, GABARAP family proteins and HeLa cell lysates to these and intrinsic full length Nd1-L, and expressing GFP-GABARAP in neurons This is an image (B) showing that Nd1-L moves to the GABARAP-positive autophagy when inducing autophagy with rapamycin.
6 is a PCR result confirming the Nd1-L gene defect (A) and the Nd1-L gene defect in the Nd1-L knockout HeLa cell prepared to confirm the function of Nd1-L (B) And Western blot results (C) showing the lack of Nd1-L protein.
7 is a Western blot result (A) showing the expression levels of autodigestion-associated proteins NBR1, p62 and GABARAPL1 in normal cells and HeLa cells lacking Nd1-L, and graphs showing quantitative analysis for proteins NBR1, p62 and GABARAPL1 ( B).
8 is an image (A) showing a decrease in the number of stress granule marker (G3BP)-GFP positive stress aggregates (SGs number) compared to normal cells when oxidative stress induction in Nd1-L-deficient HeLa cells (B) and a graph (B) )to be.

The present invention provides a screening method for a substance regulating autophagy for the prevention or treatment of degenerative nervous system diseases, comprising the steps of: (a) treating a substance to be tested on nerve cells; And (b) the Nd1-L protein present in the nucleus and cytoplasm is located as a stress aggregate, or the binding of the Nd1-L protein and GABARAP or the binding of the Nd1-L protein and GABARAPL1 is increased compared to the control group not treated with the test substance. It provides a screening method comprising the step of selecting a material to be used.

The method of screening a substance for regulating autophagy of the present invention includes a step of treating a substance to be tested on nerve cells (ie, step (a)). Step (a) is not particularly limited as long as the position of the Nd1-L protein expressed in the neuron can be changed into a stress aggregate by treating the neuron with the test substance, and the test is performed on the cultured neuron at a specific growth point. It may be a step of adding and incubating the target material.

In the method for screening an autophagy regulating substance of the present invention, compared to the control without treatment with the test substance, the Nd1-L protein present in the nucleus and cytoplasm is located as a stress aggregate, and the binding of the Nd1-L protein and GABARAP or And selecting a substance that increases the binding of the Nd1-L protein to GABARAPL1 (ie, step (b)). In step (b), the change in the position of the Nd1-L protein compared to the control group not treated with the test substance is confirmed by immunocytochemistry. In addition, the method of confirming the binding of the Nd1-L protein and the autodigestive family protein, GABARAP or GABARAPL1, may be used without limitation, as long as it is a method of confirming the interaction between the proteins, for example, immunocytochemistry or It is confirmed by a glutathione S-transferase (GST) binding assay.

In addition, the substances selected in step (b) can control abnormal accumulation of stress aggregates by activating autodigestion. The inventors of the present application confirmed that Nd1-L protein is accumulated in stress aggregates by inducing oxidation and ER stress in nerve cells in order to find out the change in the position of Nd1-L protein according to the presence or absence of stress, and accumulation of abnormal stress aggregates It was confirmed that Nd1-L protein was accumulated in stress aggregates even in neurons in which the ALS-associated mutant protein R521C was expressed.

In the structure of the Nd1-L protein, it was confirmed that the LIR (LC3-interacting region) motif sequence was present in the Kelch motif, and the binding affinity of the key proteins of the LC3 and GABARAP family proteins regulated by autodigestion As a result of the investigation, it was confirmed that the Nd1-L protein and GABARAP or the Nd1-L protein and GABARAPL1 are bound, and due to the increase of these binding, the expression of selective autodigestion related factors, NBR1, p62, and GABARAPL1, was also increased, resulting in self-digestion. It is believed that the activation of the action can control the abnormal accumulation of stress aggregates in degenerative brain diseases.

In one embodiment, the degenerative nervous system disease is a disease caused by abnormal autodigestion, and may include, for example, Lou Gehrig's disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, or frontotemporal dementia.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Experimental method and conditions

1) nerve cell culture

Neurons from E17-18 Sprague-Dawley rats (Samtako, Gyeonggi-do, Korea) were obtained from Lee et al., 2007 (Lee, JA, A. Beigneux, ST Ahmad, SG Young, and FB Gao. 2007. ESCRT- III dysfunction causes autophagosome accumulation and neurodegeneration.Curr Biol 17:1561-7.) and Lee and Gao, 2009 (Lee, JA, and FB Gao. 2009. Inhibition of autophagy induction delays neuronal cell loss caused by dysfunctional ESCRT-III in frontotemporal dementia.J Neurosci 29:8506-11.).

2) Immunocytochemistry

The neurons cultured on the plate were washed once with 1xPBS, and 400µl of 0.1% Triton-X100 was added, followed by suction after 7-10 minutes. 400 µl of 1xPBS was added and aspirated after 5 minutes, and then repeated twice. 400 µl of 3% BSA was added and blocked at room temperature for 1 hour. Thereafter, the primary antibody was diluted in 1xPBS and incubated at room temperature for 1 hour. 400 µl of 1xPBS was added and aspirated after 5 minutes, and then repeated twice, and the secondary antibody was diluted in 1xPBS. After PBS suction, 50 µl of the diluted secondary antibody was gently sprayed on the glass and incubated at room temperature for 1 hour. 400 µl of 1xPBS was added and suction was performed 5 minutes later, and then repeated twice, and mounting was performed.

3) Western Blot

Lysate obtained from cells cultured in lysis buffer (50 mM Tric-Cl (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 10% SDS and protease inhibitor) (lysate, 30-50 μg ) Using anti-Nd1-L antibody (Novus/NBP1-83180) ICC 1:100 / WB: 1:100), anti-NBR1 antibody (NBR1 (cell signaling/#9891) ICC 1:100 / WB 1: 1000), anti-p62 antibody (SQSTM1 antibody (Abnova/H00008878-M01) WB 1:50,000), anti-GABARAPL1 antibody (cell signaling/#26632) ICC 1:100 / WB 1:1000) and HRP conjugate Gated anti-mouse or anti-rabbit secondary antibody (HRP conjugated Goat anti mouse (Jackson Immuno research/115-035-146) WB 1:10,000, HRP conjugated Goat anti rabbit (Jackson Immuno research/115-035-144) WB 1 :10,000-20,000) was analyzed by Western blot.

2. Changes in Nd1-L protein location with or without stress

As a result of inducing oxidative stress by treating the cultured neurons with sodium arsenite (SA) or inducing ER stress by treating dithiothreitol (DTT), as shown in FIG. 1, Nd1 It was confirmed that the -L protein expresses G3BP-GFP (a stress aggregate protein marker) and is located as a G3BP-GFP positive stress aggregate when stress is induced.

In addition, Nd1-L accumulates in irreversible stress aggregates in neurons expressing ALS-associated mutant protein R521C, which inhibits reversible changes in stress aggregates and causes abnormal stress aggregate accumulation, and autophagy receptor NBR1 is also accumulated. It was confirmed (see Fig. 2). This means that Nd1-L is accumulated in the abnormal stress aggregates associated with the mutant protein 521C associated with ALS and plays an important role in degenerative brain diseases.

3. A study on the association of Nd1-L protein in autodigestion

It was confirmed that the Nd1-L protein has the BTB/POZ domain in the N-terminal domain and the LIR (LC3-interacting region) motif sequence in the Kelch motif (see Fig. 3), and is the core proteins of autodigestion, such as LC3 and GABARAP. The binding affinity of the family proteins was confirmed. As shown in Figure 4A, LC3, which is not located as an autophagy, a fluorescent protein with GFP attached to the GABARAP family protein, and an RFP protein attached with a 3xNLS (Nuclear localization signal) to the LIR of Nd1-L, was expressed in the LC3/GABARAP family protein. It was confirmed that GABARAP and GABARAP-L1 and Nd1-L bind. In addition, the degree of migration of each LC3 and GABARAP protein from the cytoplasm to the nucleus was quantified using the GFP fluorescence intensity (see FIG. 4B), and the binding affinity between LIR or 2xLIR and LC3/GABARAP protein was confirmed. In particular, Nd1-L 2xLIR of GABARAP, GABARAPL1 indicated that there is a binding force.

As another method of confirming the binding of the Nd1-L protein to the LC3/GABARAP protein, a Glutathione S-transferase (GST) pull-down assay using HeLa cell lysate was performed to determine the combination of LC3 and GABARAP family proteins and intracellular Nd1-L. In binding, binding of Nd1-L with GABARAP and GABARAPL1 was verified (see FIG. 5A). In addition, it was confirmed that Nd1-L present in the cells migrates to the GABARAP-positive autophagy when inducing autodigestion with rapamycin by expressing GFP-GABARAP in neurons (see FIG. 5B). In other words, it means that Nd1-L plays an important role in autophagy by binding to the autophagy binding protein GABARAP.

4. Fabrication of HeLa cell line lacking Nd1-L and verification of Nd1-L lack using the same

In order to investigate the function of the Nd1-L protein, a cell line lacking Nd1-L was constructed using the CRISPR/Cas9 system method in which guide RNA-expressing plasmid and puroPx-Cas9 plasmid were introduced into HeLa cells and selectively selected for puromycin.

First, confirm the Nd1-L targeting sequence site for the production of Guide RNA (Fig. 6A), and confirm the gene deletion in Nd1-L by Guide RNA by comparison with the wild type (WT) using Genomic PCR (Fig. 6B), and , Finally, Western blot (Fig. 6C) for the Nd1-L antibody in the Nd1-L-deficient cell line was performed to verify their lack of expression.

5. A study on the association of autodigestion using cells lacking Nd1-L

In order to investigate the association of autodigestion between normal cells and HeLa cells lacking Nd1-L, the protein expression of the autophagy receptor p62, NBR1, and the autophagy binding marker protein GABARAPL1 among the key proteins for autophagy was performed by Western blot and Nd1. It was shown that the amount of the key protein for autodigestion was significantly reduced in the cells lacking -L (see Fig. 7A), and it was very interesting in the graph that quantitatively analyzed the statistically significant autodigestion related factors (p62, NBR1 and GABARAPL1). A decrease was confirmed (see Fig. 7B).

In addition, in order to investigate whether the Nd1-L protein is involved in the formation or accumulation of stress aggregates, as shown in FIG. 8A, treatment with sodium arsenite (SA) in HeLa cells lacking Nd1-L to induce oxidative stress It was confirmed by microscopic images that the number of G3BP-GFP-positive stress aggregates significantly decreased compared to normal cells at the time, and it was confirmed that the number of stress aggregates was statistically significantly reduced by quantitative analysis and graphing (see FIG. 8B).

Overall, these results indicate that the Nd1-L protein present in the nucleus and cytoplasm in the neuron is located as a stress aggregate by stress induction, and the binding of Nd1-L protein and GABARAP or the binding of Nd1-L protein and GABARAPL1 is increased. This means that the expression of NBR1, p62, and GABARAPL1, which are related factors related to selective autophagy, is increased or decreased to regulate autodigestion, thereby controlling abnormal accumulation of stress aggregates in degenerative brain diseases.

Claims (4)

As a method for screening autophagy modulators for the prevention or treatment of degenerative nervous system diseases,
(a) treating the nerve cells with a test substance; And
(b) a screening method comprising the step of selecting a substance that increases the binding of the Nd1-L protein and GABARAP or the binding of the Nd1-L protein and GABARAPL1 compared to the control group not treated with the test substance. The screening method according to claim 1, wherein the binding of the Nd1-L protein and GABARAP or GABARAPL1 in step (b) is measured by immunocytochemistry. The screening method according to claim 1, wherein the material selected in step (b) activates autodigestion to control abnormal accumulation of stress aggregates. The screening method according to claim 1, wherein the degenerative nervous system disease is one selected from the group consisting of Lou Gehrig's disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, and frontotemporal dementia.

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