CN112426520A - Pharmaceutical composition containing ubiquitin-like modified protein and application thereof - Google Patents
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Abstract
The invention provides a pharmaceutical composition containing a ubiquitin-like modified protein and application thereof. The invention discovers that Katan p60 can be modified by SUMO, and the modified SUMO can remarkably increase the microtubule cutting level of Katan p60, promote the growth of neuron axons and further promote the repair of spinal cord injury; the invention carries out deep research on potential key sites in the SUMO modification process of the Katanin p60, and confirms that the Katanin p60 is a key protein which performs the microtubule cutting function and can be modified by SUMO, and the SUMO modification can obviously enhance the microtubule cutting capability of the Katanin p 60; the invention fully discloses the effect of SUMO modification of Katanin p60 in the processes of neuron axon growth, spinal cord injury repair and the like, confirms the phenomenon of Katanin p60 type ubiquitination modification, defines the key site for mediating the modification, discloses a new mechanism for regulating the neuron axon growth by SUMO modification of Katanin p60, and provides practical experimental evidence and scientific basis for clinical treatment of spinal cord injury.
Description
Technical Field
The invention belongs to the field of biological medicine, relates to a pharmaceutical composition containing a ubiquitin-like modified protein and application thereof, and particularly relates to a pharmaceutical composition containing the ubiquitin-like modified protein and used for promoting the growth of neuron axons and repairing spinal cord injury and application thereof.
Background
At present, repair after spinal cord injury is one of the worldwide problems, axon regeneration is the key of spinal cord injury repair, and how to regenerate damaged axons becomes a scientific hotspot. Neuronal cells belong to the highly differentiated class of cells and therefore have very limited plasticity, and regeneration of the central nervous system of adult mammals, including the spinal cord, is difficult. Current means to promote partial axonal regeneration by improving the microenvironment around damaged axons include Rho-ROC inhibition, anti-NOGO antibodies, cell transplantation, and implantation of biomaterials. However, the above means do not fully exploit the regenerative potential of axons. Microtubules, which are important for the cytoskeleton structure of neurons, are not only "trucks" for the transport of intracellular material within neurons, but are also important for the growth and development of neuronal processes. However, how intracellular and extracellular signals precisely regulate rearrangement of the intracellular frameworks of neurons has not been determined so far. Recent studies have found that microtubules and tubulin can undergo a number of post-translational modifications, and that the post-translationally modified microtubules stimulate increased sensitivity of microtubule cleaving enzymes to them, thereby modulating neurite outgrowth. Therefore, the growth and development of the neuron cells can not be separated from the modification, the dynamic property and the plasticity of microtubules, so that the regeneration of the damaged axon is also a microtubule-mediated process, and a plurality of scholars are continuously dedicated to researching the relation between the regeneration of the damaged axon and the cytoskeleton, and exploring the mechanism of the cytoskeleton from the aspect of intracellular and extracellular signals to further break through the worldwide problem of repairing the injured spinal cord.
Microtubules are an important component of maintaining the vast majority of cytoskeleton, providing structural and shape support for cells. Tubulin has a compact structure of about 8nm in length, and possesses 3 functional domains: amino N-terminal, middle domain and carboxy C-terminal, with different domains having different biological activities. The development of the nervous system is an extremely complex process supported and regulated by the neural cytoskeleton, which is composed of microtubules, actin and the intermediary filament network. And the formation and development of neuronal processes require the cutting and invasion of microtubules. Axons and dendrites develop and maintain unions from microtubules, and longer microtubules can act as "scaffolds" that prevent retraction of axons or dendrites during their development. Microtubules also play a "orbital" role in the nervous system, transporting motor proteins, targeted information to different compartments of the neuron, and regulating substances to precisely reach desired locations.
Katain is a member of the AAA cleavage protein family, which is involved in regulation of cell cycle regulation, organelle synthesis, protein vesicle transport, etc., and is capable of hydrolyzing ATP production to achieve different biological functions. As a new member of AAA family, Katanin is able to cleave microtubules, and there are four patterns of Katanin cleavage of microtubules found in the existing studies: (1) when the microtubules are combined with the dynein, the microtubules are distorted and deformed into S and V shapes, and Katanin is attracted to cut the microtubules; (2) beta microtubule content attracts katain to cut; (3) katanin identifies the branching position of the microtubules to cut the microtubules; (4) when microtubules are modified by glycosylation, glutamylation, acetylation and the like, the cutting efficiency of Katanin on microtubules is enhanced, and the cutting of Katanin can be effectively reduced by deacetylated microtubules.
The neuronal system is rich in Katanin, which promotes the passage of axonal branches by severing long microtubules and transporting them into the axon during neurite outgrowth. It is similar to the other cleavage protein, Spastin, and severely impairs axonal growth, whether knocked-down or overexpressed. The specific mechanism of Katanin in regulating neuron is not clear so far, although a few studies find that Katanin may be modified by ubiquitinated protein to regulate the growth of neuron processes, the specific mechanism is still not clear, and no knowledge is made as to whether Katanin is also regulated by other types of modification. Therefore, it is necessary to deeply research the specific mechanism of Katanin in the process of regulating the growth of neuron axons, so as to provide a new theoretical basis and medication guidance for the clinical treatment of spinal cord injury repair.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and therefore, the function and mechanism of Katan p60 in the process of regulating and controlling the growth of neuron axons are deeply researched, and the fact that Katan p60 is regulated and controlled by Ubiquitin-like modification is found, so that a key protein for mediating the modification, namely Ubiquitin-like modified protein (SUMO), is clear, a novel mechanism of the SUMO modification and control of Katan p60 in microtubule cutting and neuron axon growth is disclosed, and practical experimental evidence and scientific basis are provided for clinical treatment of spinal cord injury repair.
In order to solve the above technical problems, the present invention is achieved by the following technical solutions.
In a first aspect, the invention provides a pharmaceutical composition for promoting neuronal axonal growth and/or spinal cord injury repair, comprising a ubiquitination-like modified protein.
Preferably, the ubiquitin-like modifying protein is selected from one or more of SUMO1, SUMO2, SUMO3 and SUMO 4.
Preferably, the ubiquitination-like modified protein is SUMO 2.
The second aspect of the invention provides a composition for improving the microtubule cleavage capacity of Katanin p60, which comprises a ubiquitin-like modified protein.
Preferably, the ubiquitin-like modifying protein is selected from one or more of SUMO1, SUMO2, SUMO3 and SUMO 4.
Preferably, the ubiquitination-like modified protein is SUMO 2.
Preferably, the Katanin is selected from one or more of Katanin p60 and Katanin p 80.
Preferably, the Katanin is selected from Katanin p 60.
The third aspect of the invention provides an application of the ubiquitin-like modified protein in preparing a medicament for promoting the axon growth of neurons and/or repairing spinal cord injury.
Preferably, the ubiquitin-like modifying protein is selected from one or more of SUMO1, SUMO2, SUMO3 and SUMO 4.
Preferably, the ubiquitination-like modified protein is SUMO 2.
The fourth aspect of the invention provides an application of the ubiquitin-like modified protein in preparing a reagent for improving the Katanin microtubule cutting ability.
Preferably, the ubiquitin-like modifying protein is selected from one or more of SUMO1, SUMO2, SUMO3 and SUMO 4.
Preferably, the ubiquitination-like modified protein is SUMO 2.
Preferably, the Katanin is selected from one or more of Katanin p60 and Katanin p 80.
Preferably, the Katanin is selected from Katanin p 60.
In a fifth aspect, the invention provides the use of Katanin in the manufacture of a medicament for promoting neuronal axonal growth and/or spinal cord injury repair.
Preferably, the Katanin is selected from one or more of Katanin p60 and Katanin p 80.
Preferably, the Katanin is selected from Katanin p 60.
The SUMO modification is a protein modification mode mediated by SUMO protein, and the modification process is similar to the ubiquitination modification. There are 4 SUMO protein subtypes in mammals, SUMO1, SUMO2, SUMO3 and SUMO 4. SUMO and Ub molecules are highly similar in tertiary structure, however their physiological functions are completely different due to the large difference in surface charge. Studies have shown that basic SUMO modification can cause various neurological diseases including neurodegenerative diseases, spinocerebellar ataxia, cerebral ischemia, epilepsy and the like after being destroyed, and the role played by SUMO modification in the process of neuronal axon growth and spinal cord injury repair and the action mechanism thereof are not studied.
The inventor discovers through a large number of researches that Katanin p60 can be modified by SUMO, and the SUMO modification can remarkably enhance the microtubule cutting ability of Katanin p60, promote the growth of neuron axons and further promote the repair of spinal cord injury. The inventors of the present invention conducted intensive studies with respect to SUMO modification process, and found that Katanin p60 is a key protein that performs microtubule cleavage function and can be modified by SUMO modification at the key site of K330, and when Katanin p 60K 330 site is mutated to Katanin p 60K 330R, it results in the loss of microtubule cleavage function, and thus the growth of neurite is not promoted.
Compared with the prior art, the invention has the following technical effects:
(1) the invention discovers that Katan p60 can be modified by SUMO, and the modified SUMO can remarkably increase the microtubule cutting level of Katan p60, promote the growth of neuron axons and further promote the repair of spinal cord injury.
(2) The invention carries out intensive research on potential key sites in the SUMO modification process of Katan p60, and confirms that Katan p60 is a key protein which performs microtubule cleavage function and can be modified by SUMO, and the modified key sites are K330.
(3) The invention fully discloses the function of the Katanin p60 SUMO modification in the processes of neuron axon growth, spinal cord injury repair and the like, confirms the phenomenon of the Katanin p60 type ubiquitination modification, defines the key site for mediating the modification, discloses a new mechanism for regulating the neuron axon growth by the Katanin p60 SUMO modification, and provides practical experimental evidence and scientific basis for the clinical treatment of spinal cord injury.
Drawings
FIG. 1 shows the results of electrophoresis of purified GST-SUMO1, GST-SUMO2 and GST-SUMO 3.
FIG. 2 shows the WB detection results of GST-SUMO1/2/3 and Katanin p60 after in vitro reaction.
FIG. 3 is a schematic diagram showing the results of Katan p60 and SUMO2 co-localization detection in hippocampal neurons.
FIG. 4 is a schematic representation of the potential SUMO site of Katan p 60.
FIG. 5 is a schematic diagram of qualitative detection of the effect of mutation of potential SUMO sites of Katan p60 on microtubule cleavage function.
FIG. 6 is a schematic diagram of quantitative determination of the effect of mutation of potential SUMO sites of Katan p60 on microtubule cleavage function.
FIG. 7 is a schematic diagram showing the co-immunoprecipitation results of Katan p60-K330R and SUMO2 proteins.
FIG. 8 is a schematic diagram of qualitative detection of microtubule cleavage function by SUMO modification of Katan p 60.
FIG. 9 is a schematic diagram of quantitative determination of microtubule cleavage function by SUMO modification of Katan p 60.
FIG. 10 is a schematic representation of the effect of Katanin p60 and its mutants on hippocampal neurite outgrowth.
FIG. 11 is a graph showing the effect of Katanin p60 and its mutants on the complexity of hippocampal processes.
FIG. 12 is a graph showing the effect of Katanin p60 and its mutants on the total number of hippocampal processes.
FIG. 13 is a schematic representation of the effect of Katanin p60 and its mutants on the length of the primary processes in hippocampal neurons.
FIG. 14 is a graph showing the effect of Katanin p60 and its mutants on the overall length of hippocampal processes.
FIG. 15 is a schematic representation of the effect of SUMO modification of Katan p60 and its mutant K330R on hippocampal neurite outgrowth.
FIG. 16 is a schematic representation of the effect of SUMO modification of Katan p60 and its mutant K330R on the total number of hippocampal processes.
FIG. 17 is a schematic representation of the effect of SUMO modification of Katan p60 and its mutant K330R on the overall length of hippocampal neurons.
Detailed Description
In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Cell tools including 293T cells, rat hippocampal neurons and COS7 monkey fibroblasts listed in the context of the present invention are commercially available and cultured according to conventional methods in cell biology, unless otherwise specified. All cell lines were identified by short tandem repeat analysis of the chinese typical culture collection (wuhan) and verified for the presence of mycoplasma contamination using a PCR assay kit (shanghai Biothrive Sci) while being cryopreserved in liquid nitrogen and used for subsequent experiments. The reagents used in the present invention are commercially available. The experimental methods and techniques used in the present invention, such as COS7 culture, 293T cell culture, rat hippocampal neuron culture, Western blot, vector construction, Pulldown experiment, calcium phosphate precipitation method transgene, molecular cloning, PCR, immunofluorescence staining, laser confocal, co-immunoprecipitation, animal experiments, etc., are all conventional methods and techniques in the art.
Representative results from selection of biological experimental replicates are presented in the context figure, and data are presented as mean ± SEM as specified in the figure. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 5.0 or SPSS 20.0 software. And comparing the difference of the mean values of two or more groups by adopting a t test or one-factor analysis of variance. p < 0.05 was considered a significant difference.
Example 1 sumoylation modification of Katanin p60
(1) Constructing GST-SUMO1, GST-SUMO2 and GST-SUMO3 plasmids, and transforming the constructed plasmids into competent BL21 cells;
(2) after overnight culture, selecting a monoclonal positive colony, verifying that the colony is correct, and then taking 3 mL of bacterial liquid to culture in 250 mL of LB culture medium containing 1% ampicillin resistance, wherein the temperature of a shaking table is 37 ℃, and the rpm is 180 until the OD of the bacterial liquid is 0.8;
(3) adding 0.1mM IPTG, and inducing at 30 deg.C for 6-8 h;
(4) collecting bacterial liquid at 4 ℃, 12000rpm, adding a proper amount of lysate, carrying out ice bath for 1h, shaking for a plurality of times every 15min, and carrying out ultrasonic treatment for 6 times at the intensity of 20% in an ultrasonic crusher at 5s multiplied by 5 s;
(5) centrifuging at 4 deg.C and 12000rpm for 15min to obtain supernatant turbid supernatant;
(6) adding 200 mu L of beads into a centrifuge tube, adding 1mL of lysate, centrifuging, removing supernatant, adding turbid supernatant obtained in the step (5), and turning and combining the turbid supernatant on a 4 ℃ turning table overnight;
(7) centrifuging at 4 ℃ and 1000 rpm for 5min to remove supernatant, adding 1mL of lysate for cleaning, and repeatedly cleaning for 4 times to obtain purified protein;
(8) carrying out SDS-PAGE electrophoresis after purifying the protein, and finally carrying out Coomassie brilliant blue staining;
the result is shown in FIG. 1, the electrophoresis pattern uses the lane where the left protein Marker is located as the first lane, which is numbered from left to right as 1-8, wherein lanes 6-8 are 2 μ g, 4 μ g and 8 μ g of standard protein, respectively.
And then mixing and incubating the purified SUMO protein and rat brain lysate overnight, and then carrying out GST-Pulldown experiment, wherein the method comprises the following specific steps:
(1) weighing 400mg of rat brain tissue;
(2) extracting appropriate amount of brain lysate and adding 100 XPMSF (adding 500. mu.L cell lysate per 100mg mouse brain tissue);
(3) grinding the rat brain tissue on ice by using a grinding rod to ensure that the rat brain tissue is fully cracked;
(4) centrifuging at 12000rpm at 4 deg.C for 15min, keeping supernatant, adding 200 μ L supernatant into 5 × loading buffer, and heating with dry heat instrument at 95 deg.C for 6min to obtain positive control (Input);
(5) adding 50 mu Lbeads and 1mL of cell lysate into an EP tube, centrifugally cleaning beads, adding 500 mu L of mouse brain lysate, turning for 1h at 4 ℃, and centrifuging to take supernatant;
(6) respectively taking 50 mu g of purified protein, adding the protein into the rat brain lysate, turning over overnight at 4 ℃, centrifuging to remove supernatant, and reserving beads;
(7) washing the beads obtained in the step (6) by using Washing Buffer, adding an appropriate amount of lysate, then adding 5 × loading Buffer, heating for 6min by using a dry heat instrument, and carrying out Western Blot detection.
The results are shown in FIG. 2. The results show that GST-SUMO2 protein can be combined with Katan p60 in rat brain lysate, i.e. SUMO2 and Katan p60 can be combined with each other in vitro.
Example 2 Co-localization of Katan p60 with SUMO2 protein
(1) Culturing hippocampal neuron cells, fixing with 4% PFA at 4 deg.C for 40min, and washing with precooled PBS;
(2) perforating with TBST (containing 1% Triton X-100) at room temperature (adding 500 μ L per well of cells), 5 min/time, repeating twice;
(3) preparing 3% blocking serum (0.3 g BSA +10mL TBST), adding 200 μ L blocking serum to each cell, and blocking at room temperature for 1 h;
(4) transferring the Coverlips to an antibody incubation box, wherein the slide surface containing the cells faces upwards, washing with TBST, adding the antibody into blocking serum for dilution, dripping 60 mu L of mixed diluent of the Katanin p60 antibody and the SUMO2 antibody into each Coverlips, and incubating overnight at 4 ℃;
(5) recovering the mixed diluent of the Katanin p60 antibody and the SUMO2 antibody which are incubated in the step (4), washing with TBST, adding a secondary antibody and blocking serum, dropwise adding 60 mu L of antibody diluent into each Coverlips, and incubating for 1h at room temperature in a dark place;
(6) removing the secondary antibody diluent, washing with TBST, dropwise adding a proper amount of mounting agent on the glass slide, inversely covering the mounting agent with Coverlips, standing in a dark box, and air-drying;
(7) cells were observed and photographed under a confocal laser microscope.
As a result, as shown in FIG. 3, the position indicated by the arrow is a co-localized region of Katanin p60 and SUMO2, a yellow signal after Merge can be seen after local magnification, and the co-localized region is more obvious.
Example 3 Effect of the Katanin p60 mutant on microtubule cleavage function
(1) Constructing a GFP-Katanin p60 plasmid (NCBI Reference Sequence: NP-001004217.2);
(2) the potential 3 SUMO modification sites within Katanin p60 were determined by bioinformatics software analysis, K77, K157 and K330 respectively (see fig. 4);
(3) respectively carrying out site mutation on the GFP-Katan p60 plasmid constructed in the step (1) aiming at three sites of K77, K157 and K330 to obtain GFP-Katan p60-K77R, GFP-Katan p60-K157R, GFP-Katan p60-K330R and PAN with three sites mutated simultaneously;
(4) after COS7 cells are treated by 10mM Tubacin, COS7 cells are transfected by GFF, the GFP-Katan p60 plasmid constructed in the step (1) and the mutant plasmid obtained in the step (3);
(5) changes in Tubulin were observed by immunofluorescence chemical staining.
The inventors of the present invention found that Katan p60 has a microtubule cutting ability, and particularly has a more significant microtubule cutting effect on acetylation. For this, prior to treatment, cells were treated with Tubacin to increase the level of acetylation in microtubules. The results are shown in FIGS. 5-6, where FIG. 6 is the results of the quantitative statistical analysis of FIG. 5. The results show that overexpression of Katanin p60 clearly cleaved microtubules into small fragments compared to control GFP, whereas overexpression of the PAN full mutant plasmid did not cleave microtubules substantially, indicating that Katanin p60 lost the ability to cleave microtubules after all of the Katanin p 603 potential SUMO sites were mutated. Further experiments show that K77R and K157R do not affect microtubule cleavage, and K330R can lose microtubule cleavage capability of Katan p60, so that K330 is a SUMO site of Katan p60, can affect the cleavage capability of Katan p60, and the difference has statistical significance.
Example 4 Co-immunoprecipitation experiment of Katan p60-K330R and SUMO2 protein
(1) Respectively constructing HA-SUMO2, GFP-Katan p60, GFP-Katan p60-K330R and Flag-ubc9 plasmids;
(2) respectively transfecting the plasmids constructed in the step (1) into 293T cells;
(3) after culturing for 48 hours, adding cell lysis solution to lyse cells and collecting cell supernatant;
(4) adding 200 mu L of cell supernatant into an EP tube containing beads, turning the tube at 4 ℃ for 1h, and then keeping the supernatant;
(5) adding an HA antibody into the supernatant obtained in the step (4), and performing turnover incubation at 4 ℃ overnight;
(6) adding the solution obtained in the step (5) into an EP tube containing beads pre-washed by cell lysate, turning over for 3 hours at 4 ℃, centrifuging and removing supernatant;
(7) adding 20 μ L cell lysate and 5 × loading buffer, and heating with a dry heat instrument at 95 deg.C for 6 min;
(8) western Blot was performed to verify whether the co-immunoprecipitate product contained GFP-Katan p 60.
The results are shown in FIG. 7, and show that the wild type of Katan p60 can interact with SUMO2 protein, while Katan p60-K330R cannot bind to SUMO 2. Further proves that K330 is a key site for SUMO2 binding, and after mutation at the site, Katan p60 loses the ability of binding with SUMO2 and cannot be modified by ubiquitin-like modification, thereby losing the function of cutting microtubules.
Example 5 Effect of SUMO modification of Katan p60 on its microtubule cleavage function
(1) Constructing mCherry-SUMO2 plasmid of rat SUMO enzyme;
(2) treating COS7 cells with 10mM Tubacin, co-transforming the plasmid constructed in the step (1) with GFP-Katan p60 plasmid and Katan p60-K330R plasmid to COS7 cells, and expressing for 24 h;
(3) COS7 cells were fixed with 4% PFA at 4 deg.C for 40min, and washed with pre-cooled PBS;
(4) perforating with TBST (adding 500. mu.L per well of cells) at room temperature for 5 min/time, repeating twice;
(5) preparing 3% blocking serum (0.3 g BSA +10 mLTBST), adding 200 μ L blocking serum into each hole cell, and blocking for 1h at room temperature;
(6) transferring the Coverlips to an antibody incubation box with the cell-containing slide facing up, washing with TBST, adding blocking serum to the Tubulin antibody for dilution, adding 60 mu LTubulin antibody diluent dropwise to each Coverlips, and incubating overnight at 4 ℃;
(7) recovering the Tubulin antibody diluent incubated in the step (6), washing with TBST, adding a secondary antibody and blocking serum, dropwise adding 60 mu L of antibody diluent into each Coverlips, and incubating for 1h at room temperature in a dark place;
(8) removing the secondary antibody diluent, washing with TBST, dropwise adding a proper amount of mounting agent on the glass slide, inversely covering the mounting agent with Coverlips, standing in a dark box, and air-drying;
(9) cells were observed and photographed.
The results are shown in FIGS. 8-9, where FIG. 9 is the quantitative assay of FIG. 8pLess than 0.05, with statistical significance, and the scale size of 20 μm. The results show that overexpression of Katanin p60 caused microtubule cleavage, which was cleaved into fragments of varying length, compared to the GFP control; after the Katan p60 and the SUMO2 are co-rotated, the microtube cutting capability of the Katan is remarkably enhanced, and the microtubes are basically cut and crushed; however, after the Katan p 60K 330R and SUMO2 are co-transformed, K330, which is the key site for realizing SUMO formation by Katan p60, is mutated, so that the K330 cannot be combined with SUMO2, and further ubiquitin-like modification cannot be realized, and finally microtubule cutting ability is completely lost.
Example 6 Effect of Katanin p60 and its mutants on hippocampal neurites
(1) Culturing and observing hippocampal neuron cells, and paying attention to the state of the hippocampal neuron cells and whether the hippocampal neuron cells are polluted or not;
(2) transferring the original culture medium to auxiliary wells, and adding 500 mu L of Neurobasal culture medium into each well for starvation;
(3) using the calcium phosphate transfection kit, 1. mu.g of GFP, GFP-Katanin p60 and its mutant plasmid, and 25. mu.L of CaCl were added to the EP tube2Adding another EP tube into BBS solution;
(4) mixing the solution in the step (3), and standing for 15min in a dark box;
(5) dripping 50 μ L of mixed solution into each well, dripping the mixed solution into the wells in different directions, gently shaking for several times, after 40min, cleaning the calcium-phosphorus particles with 1 × SA for 2 times, and adding the original culture medium;
(6) after 48h of transfection, cells were fixed with 4% PFA, immunofluorescent-chemical stained and visualized under laser confocal.
The results are shown in FIGS. 10-14, whereinpLess than 0.05, with statistical significance, and the scale size of 20 μm. The results showed that the neuronal processes of GFD-Katan p60, GFP-Katan p60-K77R and GFP-Katan p60-K157R all had significantly increased complexity, total number of processes, primary process length and total length of processes relative to the blank control group GFP; the GFP-Katan p60-PAN and GFP-Katan p60-K330R have no obvious difference relative to the control group.
Further, GFP-Katan p60, GFP-Katan p60-K330R were selected to cotransform neurons with SUMO2, respectively, and the results of the experiments on the effect of neurite outgrowth were observed, as shown in FIGS. 15 to 17. The results show that co-expression of GFP-Katan p60WT and SUMO2 can further promote shoot growth, while there is no promotion if GFP-Katan p60-K330R and SUMO2 are overexpressed.
By combining the results, the invention discovers that the Katanin p60 can be modified by SUMO, and the modified SUMO can remarkably increase the microtubule cutting ability of Katanin p60, promote the growth of neuron axons and further promote the repair of spinal cord injury; further, the invention carries out deep research on potential key sites in the SUMO modification process of the Katanin p60, and confirms that the Katanin p60 is a key protein which performs the microtubule cleavage function and can be modified by SUMO, and the SUMO modification can obviously enhance the microtubule cleavage capability of the Katanin p 60; the key site of the modification is K330, and after the site is mutated, Katan p60 loses the ability of combining with SUMO2 and cannot be modified by ubiquitin-like, so that the function of cutting microtubules is lost; the invention fully discloses the effect of SUMO modification of Katanin p60 in the processes of neuron axon growth, spinal cord injury repair and the like, confirms the phenomenon of Katanin p60 type ubiquitination modification, defines the key site for mediating the modification, discloses a new mechanism for regulating the neuron axon growth by SUMO modification of Katanin p60, and provides practical experimental evidence and scientific basis for clinical treatment of spinal cord injury.
The above detailed description section specifically describes the analysis method according to the present invention. It should be noted that the above description is only for the purpose of helping those skilled in the art better understand the method and idea of the present invention, and not for the limitation of the related contents. The present invention may be appropriately adjusted or modified by those skilled in the art without departing from the principle of the present invention, and the adjustment and modification also fall within the scope of the present invention.
Claims (10)
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| DEBASISH KUMAR GHOSH等: "Models, Regulations, and Functions of Microtubule Severing by Katanin", 《ISRN MOLECULAR BIOLOGY》 * |
| KEEN CHEN等: "Katanin p60 promotes neurite growth and collateral", 《INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE》 * |
| 纪志盛: "Spastin的SUMO修饰介导微管动力调控海马神经元生长发育", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
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