High affinity chimeric polypeptide sensor and ubiquitin detection methods and kits

Abstract

This invention discloses a high-affinity chimeric peptide sensor and a ubiquitin detection method and kit, belonging to the field of biodetection technology. To address the problems of low affinity and poor selectivity in existing ubiquitin detection tools, this invention obtains a V1091L mutant of the HDAC6 ZnF fragment through multidimensional mutagenesis, and integrates other ubiquitin-binding domains using AI and computer simulation to construct a chimeric peptide sensor. Its Kd with monoubiquitin is less than 50 nM, and it can specifically bind some free ubiquitin. This invention also provides a detection method based on this sensor, enabling the detection of free ubiquitin and total ubiquitin through direct or competitive assays. It can calculate the amount of conjugated ubiquitin, determine deubiquitinase activity, and screen ubiquitin pathway regulators. A kit incorporating this sensor is also provided. The sensor of this invention exhibits high affinity and high selectivity, and the detection method requires no complex instruments, making it suitable for complex biological samples and high-throughput detection. It can also distinguish intact free ubiquitin in serum, providing a new tool for basic ubiquitin research and clinical detection, and has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a high-affinity ubiquitin-binding chimeric peptide sensor, a method for detecting ubiquitin (especially free ubiquitin) in samples using the chimeric peptide sensor, and a detection kit containing the chimeric peptide. This invention can also be applied to scenarios such as deubiquitinase activity assay, screening of ubiquitin pathway regulators, and clinical serum ubiquitin detection. Background Technology

[0002] Ubiquitin is a highly conserved small protein in eukaryotic cells that, along with ubiquitin-like proteins, regulates various cellular processes such as protein degradation, DNA repair, and cell cycle control. It exists in two forms: anchored ubiquitin (conjugated to target proteins) and free ubiquitin (not attached to target proteins, with no C-terminus conjugated). Free ubiquitin plays a crucial role in aggregate disintegration, viral infection regulation, and interferon expression, and is closely related to neurodegenerative diseases and viral infections. However, current research on the function of free ubiquitin faces significant knowledge gaps due to limitations in detection tools.

[0003] Existing tools for detecting free ubiquitin are mainly based on the BUZ domain of isoubiquitinase T. Related methods include absolute quantification of protein standards using mass spectrometry and visualization of IsoT-BUZ-derived proteins using fused fluorophores. However, these methods suffer from low affinity, poor enrichment efficiency, inaccurate quantification, and lack of binding selectivity. Furthermore, the few existing methods capable of detecting intact free C-terminal ubiquitin are unsuitable for complex biological samples such as cell or tissue lysates, require the use of radioactive ATP, and do not support high-throughput detection, making them difficult for most laboratories to implement.

[0004] Therefore, there is an urgent need to develop a high-affinity, high-selectivity free ubiquitin detection tool that can be applied to complex biological samples and achieve high-throughput detection, as well as supporting detection methods and kits, to fill the gaps in existing technologies and promote the development of basic research and clinical testing related to ubiquitin. Summary of the Invention

[0005] The purpose of this invention is to address the technical problems of existing ubiquitin detection tools, such as low affinity and poor selectivity for free ubiquitin, unsuitability for complex biological samples, inability to perform high-throughput detection, and complex operation. This invention provides a high-affinity chimeric peptide sensor, along with a ubiquitin detection method and kit based on this sensor, enabling specific detection of free ubiquitin and accurate detection of total ubiquitin. It can also be used for the determination of deubiquitinase activity and the screening of ubiquitin pathway regulators.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A chimeric polypeptide capable of binding monoubiquitin with a dissociation constant Kd of less than 50 nM, the dissociation constant being assessed by isothermal titration calorimetry (ITC); the chimeric polypeptide comprises a mutant amino acid sequence of mouse HDAC6 extended zinc finger ZnF domain 1007-1149, the mutant amino acid sequence being a V1091L mutant in which valine at position 1091 of the domain is replaced by leucine.

[0007] Furthermore, it also contains an amino acid sequence of bovine Rabex-5 ubiquitin-binding zinc finger or an amino acid sequence of ubiquitin-interacting motifs of Saccharomyces cerevisiae VPS27 or mouse RAP80, said amino acid sequence being linked to the C-terminus of the V1091L mutant via a linker; this chimeric polypeptide is capable of binding monoubiquitin with a dissociation constant Kd of less than 20 nM.

[0008] Furthermore, it can selectively bind to free ubiquitin, which includes free ubiquitin monomers and free polyubiquitin.

[0009] An isolated nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding a chimeric polypeptide as described in any one of claims 1-3.

[0010] A method for detecting or measuring ubiquitin levels in a sample, comprising the step of contacting the sample with a chimeric polypeptide as described in any one of claims 1-3; using the chimeric polypeptide as described in claim 1 or 2 to detect total ubiquitin levels, and using the chimeric polypeptide as described in claim 3 to detect free ubiquitin levels, wherein the amount of conjugated ubiquitin is calculated by subtracting the amount of free ubiquitin from the amount of total ubiquitin levels.

[0011] A method for determining the activity of a known or candidate deubiquitinase includes the following steps: co-incubating a sample containing conjugated ubiquitin with the known or candidate deubiquitinase, then contacting the incubated sample with the chimeric polypeptide of claim 3, detecting and measuring the amount of free ubiquitin in the sample, and determining the activity of the deubiquitinase based on the change in the amount of free ubiquitin.

[0012] A method for identifying agents that regulate ubiquitin-substrate conjugation, comprising the following steps: a) Contact and incubate a mixture containing ubiquitin and ubiquitin substrate with the candidate drug; b) Contact the reaction product of step a with the chimeric polypeptide of claim 3; c) Detect and determine the amount of free ubiquitin bound to the chimeric peptide, and identify whether the candidate drug is a ubiquitin conjugation modulator based on changes in the amount of free ubiquitin.

[0013] Furthermore, the sample is a biological sample, including one or more of blood, serum, cells, tissue, biopsy samples, urine, or cell lysate.

[0014] A kit for measuring ubiquitin in a sample, comprising the chimeric polypeptide of any one of claims 1-3, or the isolated nucleic acid molecule of claim 4.

[0015] Furthermore, it also includes one or more of ubiquitin standards, detectable labeling reagents, incubation buffers, and washing solutions, wherein the detectable labeling reagents include one or more of fluorophores, radionuclides, fluorescent proteins, or enzymes.

[0016] Compared with the prior art, the present invention has the following significant advantages: 1. The chimeric peptide sensor of the present invention has a high binding affinity for ubiquitin. The sensor that specifically binds to free ubiquitin can accurately distinguish between free ubiquitin and anchored ubiquitin, thus solving the problems of low affinity and poor selectivity of existing tools. 2. The ubiquitin detection method of the present invention does not require the use of complex instruments and reagents such as high performance liquid chromatography, mass spectrometry, heavy isotope standards, antibodies or gel electrophoresis, and does not require radioactive ATP. It is simple to operate and can be implemented in most laboratories. 3. The detection method of the invention is applicable to complex biological samples such as blood, serum, cells, and tissue lysates, and can realize in vitro / ex vivo quantitative detection, and supports high-throughput detection in 96-well plates; 4. The sensor, detection method, and kit of the present invention can realize real-time quantitative determination of deubiquitinase activity and can also be used for high-throughput screening of ubiquitin pathway regulators, providing tools for basic research related to ubiquitin. 5. The detection method of the present invention can distinguish between intact free ubiquitin and partially degraded free ubiquitin in serum, enabling accurate detection of serum ubiquitin in clinical settings and providing a basis for clinical assessment of stress, trauma and related diseases. Attached Figure Description

[0017] Figure 1 The graph shows the affinity maturation of the HDAC6 ZnF domain and the binding affinity determination of the V1091L mutant with monoubiquitin. Figure 2 Computer-aided design process for chimeric ubiquitin-binding proteins (UBPs) and structural design and monoubiquitin binding affinity determination of representative chimeric peptides (UBP10 / UBP05); Figure 3 This is a verification diagram of the selective enrichment of free ubiquitin by UBP10. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0019] A chimeric polypeptide capable of binding monoubiquitin with a dissociation constant Kd of less than 50 nM, the dissociation constant being assessed by isothermal titration calorimetry (ITC); the chimeric polypeptide comprises a mutant amino acid sequence of mouse HDAC6 extended zinc finger ZnF domain 1007-1149, the mutant amino acid sequence being a V1091L mutant in which valine at position 1091 of the domain is replaced by leucine.

[0020] Furthermore, it also contains an amino acid sequence of bovine Rabex-5 ubiquitin-binding zinc finger or an amino acid sequence of ubiquitin-interacting motifs of Saccharomyces cerevisiae VPS27 or mouse RAP80, said amino acid sequence being linked to the C-terminus of the V1091L mutant via a linker; this chimeric polypeptide is capable of binding monoubiquitin with a dissociation constant Kd of less than 20 nM.

[0021] Furthermore, it can selectively bind to free ubiquitin, which includes free ubiquitin monomers and free polyubiquitin.

[0022] An isolated nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding a chimeric polypeptide as described in any one of claims 1-3.

[0023] A method for detecting or measuring ubiquitin levels in a sample, comprising the step of contacting the sample with a chimeric polypeptide as described in any one of claims 1-3; using the chimeric polypeptide as described in claim 1 or 2 to detect total ubiquitin levels, and using the chimeric polypeptide as described in claim 3 to detect free ubiquitin levels, wherein the amount of conjugated ubiquitin is calculated by subtracting the amount of free ubiquitin from the amount of total ubiquitin levels.

[0024] A method for determining the activity of a known or candidate deubiquitinase includes the following steps: co-incubating a sample containing conjugated ubiquitin with the known or candidate deubiquitinase, then contacting the incubated sample with the chimeric polypeptide of claim 3, detecting and measuring the amount of free ubiquitin in the sample, and determining the activity of the deubiquitinase based on the change in the amount of free ubiquitin.

[0025] A method for identifying agents that regulate ubiquitin-substrate conjugation, comprising the following steps: a) Contact and incubate a mixture containing ubiquitin and ubiquitin substrate with the candidate drug; b) Contact the reaction product of step a with the chimeric polypeptide of claim 3; c) Detect and determine the amount of free ubiquitin bound to the chimeric peptide, and identify whether the candidate drug is a ubiquitin conjugation modulator based on changes in the amount of free ubiquitin.

[0026] Furthermore, the sample is a biological sample, including one or more of blood, serum, cells, tissue, biopsy samples, urine, or cell lysate.

[0027] A kit for measuring ubiquitin in a sample, comprising the chimeric polypeptide of any one of claims 1-3, or the isolated nucleic acid molecule of claim 4.

[0028] Furthermore, it also includes one or more of ubiquitin standards, detectable labeling reagents, incubation buffers, and washing solutions, wherein the detectable labeling reagents include one or more of fluorophores, radionuclides, fluorescent proteins, or enzymes.

[0029] Specifically: This invention employs multidimensional mutagenesis to select and mature the HDAC6 ZnF fragment, resulting in a mutant with significantly enhanced binding affinity. Specifically, valine at position 1091 of the mouse HDAC6 extended zinc finger domain (1007-1149) is replaced with leucine (V1091L). Furthermore, using artificial intelligence tools and computer simulations, the de novo designed structure is integrated with known ubiquitin-binding domains such as the bovine Rabex-5 ubiquitin-binding zinc finger, Saccharomyces cerevisiae VPS27, or mouse RAP80 ubiquitin-binding motif into this mutant. Domain connection is achieved through linkers, yielding a high-affinity chimeric peptide sensor.

[0030] This chimeric peptide sensor can selectively bind free ubiquitin, or simultaneously bind free and conjugated ubiquitin / ubiquitin-like proteins. Its dissociation constant (Kd) with monoubiquitin is less than 50 nM, and after integrating additional ubiquitin-binding domains, Kd can be less than 20 nM. The chimeric peptide contains at least one linker, which consists of 2 to 30 amino acids, mainly glycine, serine, alanine, or cysteine. Its length and flexibility can be adapted to accommodate the simultaneous binding of multiple ubiquitin-binding domains to different regions of ubiquitin.

[0031] This invention also provides a ubiquitin detection method based on the above-mentioned chimeric peptide sensor. By contacting the sample with the chimeric peptide sensor and directly measuring or indirectly competing to measure, the content of free ubiquitin and total ubiquitin in the sample can be detected. The content of conjugated ubiquitin can be calculated as conjugated ubiquitin = total ubiquitin - free ubiquitin. This method can be used for the quantitative and real-time determination of deubiquitinase (DUB) activity. By comparing the free ubiquitin content in samples with and without DUB, or by monitoring the changes in free ubiquitin at different time points, DUB activity can be determined. At the same time, by detecting changes in the content of free ubiquitin in candidate drugs, agents that regulate the conjugation of ubiquitin with substrates can be screened.

[0032] The present invention further provides a ubiquitin detection kit, which includes the above-mentioned chimeric polypeptide sensor or a separated nucleic acid molecule encoding the chimeric polypeptide, and can be directly used for the detection of ubiquitin in various biological samples.

[0033] The following detailed description is based on the technical solution of the present invention. The scope of protection of the present invention is not limited to the following embodiments. Equivalent substitutions made by those skilled in the art for the technical features of the present invention are all within the scope of protection of the present invention.

[0034] Construction of chimeric peptide sensors Mutagenesis and affinity maturation of HDAC6 ZnF fragment: Mutagenesis of mouse HDAC6 extended zinc finger (ZnF) domains 1007-1149 was performed using multidimensional mutagenesis techniques to screen for affinity-mature mutants. In this mutant, valine at position 1091 was replaced with leucine (V1091L). The dissociation constant (Kd) of this mutant with monoubiquitin was less than 50 nM, and its binding affinity could be verified by isothermal titration calorimetry (ITC).

[0035] Integration of ubiquitin-binding domains: Using artificial intelligence tools and computer simulations, the Ruz domain of bovine Rabex-5, the UIM domain of Saccharomyces cerevisiae VPS27, or the UIM domain of mouse RAP80 were integrated into the C-terminus of the V1091L mutant. The domains were connected by linkers. The Kd of the integrated chimeric peptide with monoubiquitin can be less than 20 nM and can specifically bind free ubiquitin.

[0036] Linker design: The linker is a linear peptide chain of 2 to 30 amino acids, mainly composed of glycine, serine, alanine or cysteine. One or two linkers can be designed according to the number of ubiquitin-binding domains. The length and flexibility of the linker must ensure that each ubiquitin-binding domain can simultaneously contact and bind to non-overlapping regions of ubiquitin, such as the ubiquitin C-terminus and surface hydrophobic patches.

[0037] In the chimeric polypeptide of the present invention, the sequence identity of the ubiquitin-binding domain with that of a known ubiquitin-binding domain is 75% to 100%, and the sequence identity of the bound ubiquitin protein with that of human ubiquitin protein is at least 80%, preferably human ubiquitin protein; a detectable labeled binding site can be introduced on the ubiquitin protein by amino acid deletion, addition or substitution, such as replacing the 20th serine of ubiquitin with cysteine ​​(S20C).

[0038] Sample processing for ubiquitin detection The samples to be tested are biological samples, including blood, serum, cells, tissues, biopsy samples, urine or cell lysates, etc. Solid or semi-solid samples are lysed and centrifuged, and the supernatant is used as the test sample. Liquid samples can be tested directly or diluted and purified as needed.

[0039] ubiquitin assay based on chimeric peptide sensor Direct titration determination: The chimeric peptide sensor with detectable label is contacted with ubiquitin standards of gradient concentration and the sample to be tested, respectively. After incubation, the signal changes of the sensor label (such as fluorescence intensity and fluorescence anisotropy) are measured, a standard curve is plotted, and the ubiquitin content in the sample is calculated based on the standard curve. Sensors that bind only free ubiquitin can be selected to detect free ubiquitin, and sensors that bind both free and conjugated ubiquitin can be selected to detect total ubiquitin.

[0040] Competitive assay: A ubiquitin competitor with a detectable label is pre-bound to a chimeric peptide sensor, and then the sample to be tested is added. The ubiquitin in the sample will compete with the competitor for the binding site of the sensor. After incubation, the signal change of the competitor label is measured, and the ubiquitin content in the sample is calculated based on the degree of signal change. Detectable labels include fluorescent proteins such as fluorophores, radionuclides, enzymes, GFP, and mCherry. Detection methods include fluorescence intensity, fluorescence anisotropy measurement, or FRET technology.

[0041] Calculation of conjugated ubiquitin: The sample to be tested is divided into two equal parts. The free ubiquitin content of one part is detected by a sensor that binds only free ubiquitin, and the total ubiquitin content of the other part is detected by a sensor that binds both free and conjugated ubiquitin. The conjugated ubiquitin content is calculated by the formula: conjugated ubiquitin content = total ubiquitin content - free ubiquitin content.

[0042] Assay for deubiquitinase (DUB) activity Control method: An experimental group and a negative control group were set up. The experimental group was incubated with the DUB to be tested and the conjugated ubiquitin substrate, while the negative control group was incubated with the conjugated ubiquitin substrate only. After incubation, the free ubiquitin content of the two groups was detected by the chimeric peptide sensor of the present invention. If the free ubiquitin content of the experimental group was significantly higher than that of the negative control group, it indicated that the DUB to be tested had deubiquitinase activity, and the content difference was positively correlated with the DUB activity.

[0043] Real-time monitoring method: DUB is co-incubated with conjugated ubiquitin substrate, and samples are taken at different time points. The free ubiquitin content at each time point is detected by a chimeric peptide sensor. If the free ubiquitin content gradually increases with time, it indicates that the DUB to be tested has deubiquitinase activity, and the rate of change in content is positively correlated with the DUB activity.

[0044] High-throughput screening of ubiquitin pathway regulators Ubiquitin, ubiquitin substrates, and candidate drugs were co-incubated, with a blank control group (without candidate drugs) included. After incubation, the free ubiquitin content in each group was detected using a chimeric peptide sensor that binds only free ubiquitin. If the free ubiquitin content in the candidate drug group was higher than that in the blank control group, it indicated that the drug could inhibit ubiquitin conjugation with the substrate or possess deubiquitinase activity; if the free ubiquitin content in the candidate drug group was lower than that in the blank control group, it indicated that the drug could promote ubiquitin conjugation with the substrate or inhibit deubiquitinase activity. This screening can be performed in 96-well plates, enabling high-throughput screening of candidate drugs such as small molecules, peptides, or polypeptides.

[0045] Clinical serum ubiquitin detection Patient serum samples are collected, and the content of free ubiquitin in the serum is detected using the chimeric polypeptide sensor that binds to the C-terminus of ubiquitin in this invention. This sensor binds only to free ubiquitin with an intact C-terminus, and can distinguish between intact free ubiquitin and partially degraded free ubiquitin. Combined with changes in serum ubiquitin levels, the patient's stress and trauma status can be assessed, providing a basis for the clinical diagnosis and assessment of related diseases.

[0046] Composition and use of the test kit The ubiquitine detection kit of the present invention comprises the core components: the chimeric polypeptide sensor described in the present invention (which may be selected to bind only free ubiquitine or to bind both free and conjugated ubiquitine), or the isolated nucleic acid molecule encoding the chimeric polypeptide; the kit may also contain auxiliary reagents such as ubiquitine standards, detectable labeling reagents, incubation buffer, and washing solution.

[0047] Instructions for use of the kit: Following the kit instructions, mix the chimeric peptide sensor with the sample to be tested in the specified ratio and incubate. Detect signal changes by direct titration or competitive assay. Calculate the content of free ubiquitin or total ubiquitin in the sample based on the standard curve. The operation is simple and enables rapid in vitro detection. Industrial applicability

[0048] The chimeric peptide sensor of this invention can be prepared on a large scale through genetic engineering technology. The detection method based on this sensor is simple to operate and accurate. The matching kit can realize standardized production and commercial application. The product and method of this invention can be applied to basic research on ubiquitin in biological laboratories, screening of ubiquitin pathway regulators in drug development companies, and clinical serum ubiquitin detection in medical institutions, and has significant industrial application value.

[0049] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A chimeric polypeptide, characterized in that, It can bind monoubiquitin with a dissociation constant Kd of less than 50 nM, which was assessed by isothermal titration calorimetry (ITC). The chimeric polypeptide contains a mutant amino acid sequence of mouse HDAC6 extended zinc finger ZnF domain 1007-1149, wherein the mutant amino acid sequence is a V1091L mutant in which valine at position 1091 of the domain is replaced with leucine.

2. The chimeric polypeptide according to claim 1, characterized in that, It also contains an amino acid sequence of bovine Rabex-5 ubiquitin-binding zinc finger or an amino acid sequence of ubiquitin-interacting motifs of Saccharomyces cerevisiae VPS27 or mouse RAP80, said amino acid sequence being linked to the C-terminus of the V1091L mutant via a linker; the chimeric polypeptide is capable of binding monoubiquitin with a dissociation constant Kd of less than 20 nM.

3. The chimeric polypeptide according to claim 1 or 2, characterized in that, It can selectively bind to free ubiquitin, which includes free ubiquitin monomers and free polyubiquitin.

4. An isolated nucleic acid molecule, characterized in that, It comprises or consists of a nucleic acid sequence encoding the chimeric polypeptide of any one of claims 1-3.

5. A method for detecting or measuring ubiquitin levels in a sample, characterized in that, The method includes the step of contacting the sample with the chimeric polypeptide described in any one of claims 1-3; using the chimeric polypeptide described in claim 1 or 2 to detect the total ubiquitin content, using the chimeric polypeptide described in claim 3 to detect the free ubiquitin content, wherein the amount of conjugated ubiquitin is calculated by subtracting the amount of free ubiquitin from the total ubiquitin content.

6. A method for determining the activity of known or candidate deubiquitinases, characterized in that, Includes the following steps: The sample containing conjugated ubiquitin is co-incubated with the known or candidate deubiquitinase, and then the incubated sample is contacted with the chimeric polypeptide of claim 3. The amount of free ubiquitin in the sample is detected and measured, and the activity of the deubiquitinase is determined based on the change in the amount of free ubiquitin.

7. A method for identifying agents that regulate ubiquitin-substrate conjugation, characterized in that, Includes the following steps: a) Contact and incubate a mixture containing ubiquitin and ubiquitin substrate with the candidate drug; b) Contact the reaction product of step a with the chimeric polypeptide of claim 3; c) Detect and determine the amount of free ubiquitin bound to the chimeric peptide, and identify whether the candidate drug is a ubiquitin conjugation modulator based on changes in the amount of free ubiquitin.

8. The method according to any one of claims 5-7, characterized in that, The sample is a biological sample, including one or more of blood, serum, cells, tissue, biopsy samples, urine, or cell lysate.

9. A kit for measuring ubiquitin in a sample, characterized in that, It comprises the chimeric polypeptide of any one of claims 1-3, or the isolated nucleic acid molecule of claim 4.

10. The reagent kit according to claim 9, characterized in that, It also contains one or more of ubiquitin standards, detectable labeling reagents, incubation buffers, and washing solutions, wherein the detectable labeling reagents include one or more of fluorophores, radionuclides, fluorescent proteins, or enzymes.

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