CN-121991919-A - PK5a1 hidden exon and protein product N207 marker and application thereof

CN121991919ACN 121991919 ACN121991919 ACN 121991919ACN-121991919-A

Abstract

A PK5a1 hidden exon and protein product N207 marker and application thereof belong to the technical field of molecular medicine, wherein about 95% of amyotrophic lateral sclerosis ALS groups and about 57% of Alzheimer disease AD groups have TDP-43 pathological abnormalities, which indicate that the TDP-43 pathological abnormalities are important molecular mechanisms of two diseases, and further experiments prove that the loss or the knockdown of the TDP-43 function can directly induce the activation of the PK5a1 hidden exon, which indicates that the generation of the PK5a1 depends on the TDP-43 pathological mechanisms. On this basis, the present invention detects PK5a1 activation in brain tissues, iPSC neurons and animal models of ALS and AD groups, and stably detects the translational product N207 protein in cerebrospinal fluid and blood samples. The biomarker can be used as a detection target for pre-ALS AD occurrence or used as a target for preparing medicines for treating and/or preventing related neurodegenerative diseases.

Inventors

YANG MINGMING
WANG QI
Gui Yuran
HUANG ZHIZHOU
WANG XIAOCHUAN

Assignees

江汉大学

Dates

Publication Date: 20260508
Application Date: 20251229

Claims (7)

1. A PK5a1 hidden exon and a protein product N207 marker are characterized in that the nucleic acid sequence of the PK5a1 hidden exon is shown as SEQ ID NO.1 and ：GACTGGCCCTGTGAGTGATGGTGTTTGGATAAAAGAGTCTGGGTGGAAGTGTGGCTGCCTGCAGGTGACAGTGCGACAGTGTCACTGTGCCTGCCCCTTGGATGAAGCGTGATGCATCTTCATGATG,, and the amino acid sequence of the protein product N207 of the PK5a1 hidden exon is shown as SEQ ID NO.2 ：MASDAVQSEPRSWSLLEQLGLAGADLAAPGVQQQLELERERLRREIRKELKLKEGAENLRRATTDLGRSLGPVELLLRGSSRRLDLLHQQLQELHAHVVLPDPAATHDGPQSPGAGGPTCSATNLSRVAGLEKQLAIELKVKQGAENMIQTYSNGSTKDRKLLLTAQQMLQDSKTKIDIIRMQLRRALQAGQLENQAAPDDTQGLAL*.
2. Use of the PK5a1 harboring exon and protein product N207 marker according to claim 1 in the preparation of a kit for detecting amyotrophic lateral sclerosis ALS.
3. The use according to claim 2, wherein the detection method of the kit for detecting amyotrophic lateral sclerosis ALS is qRT-PCR, RNA sequencing, immunoblotting, ELISA or mass spectrometry detection.
4. Use of the PK5a1 harboring exon and protein product N207 marker according to claim 1 in ALS and AD risk prediction systems.
5. The use of claim 4, wherein the ALS and AD risk prediction system steps are: Firstly, extracting RNA or protein by a sample pretreatment module; secondly, detecting PK5a1 or N207 level by a molecular detection module; And thirdly, a data analysis module is used for establishing a prediction model by combining clinical indexes.
6. The method according to claim 5, wherein the clinical index is ALSFRS-R and/or the level of neurofilament light chain NEFL.
7. The method according to claim 2 to 6, wherein the PK5a1 activation is dependent on a TDP-43 deficiency, and wherein the TDP-43 knock-down induces the production of the hidden exon of PK5a 1.

Description

PK5a1 hidden exon and protein product N207 marker and application thereof Technical Field The invention belongs to the technical field of molecular medicine, and particularly relates to a PK5a1 hidden exon and a protein product N207 marker and application thereof. Background ALS is a fatal neurodegenerative disease with an average survival of only 3-5 years, and AD is the most common dementia disease worldwide. Both are clinically distinct but manifest as progressive dysfunction and death of neurons. Previous pathology studies showed that TDP-43 pathology, including loss of nuclear function and cytoplasmic aggregate deposition, was present in about 95% of ALS groups and about 57% of AD groups. This phenomenon suggests that TDP-43 pathology is a key molecular mechanism for both types of disease. However, there is currently a lack of molecular markers that can directly reflect TDP-43 dysfunction. Clinically common biomarkers include body fluid indexes such as neurofilament light chain (NEFL) and Glial Fibrillary Acidic Protein (GFAP), but the sensitivity and the specificity are limited, so that the activation of a hidden exon caused by the defect of TDP-43 function is difficult to reflect, and the transcriptome disorder of ALS and AD cannot be fully explained. Therefore, it is highly desirable to find a molecular marker that truly reflects the pathological functional defects of TDP-43 and is detectable in ALS and AD groups to enable the preparation of kits for early diagnosis of disease, risk prediction and disease course monitoring. Disclosure of Invention The application aims to solve the technical problems that the existing prior art lacks a molecular marker capable of directly reflecting TDP-43 dysfunction, a common biomarker is difficult to reflect hidden exon activation caused by TDP-43 dysfunction, the transcriptome disorder of ALS and AD cannot be fully explained, and the like, and provides a PK5a1 hidden exon and protein product N207 marker and application thereof. The invention aims to systematically identify PKN1 gene hidden exon PK5a1 and a translation product N207 protein thereof for the first time, and the protein is used as a specific molecular marker of neurodegenerative diseases. Technical proposal is that a PK5a1 hidden exon and a protein product N207 marker are provided, the nucleic acid sequence of the PK5a1 hidden exon is shown as SEQ ID NO.1 and ：GACTGGCCCTGTGAGTGATGGTGTTTGGATAAAAGAGTCTGGGTGGAAGTGTGGCTGCCTGCAGGTGACAGTGCGACAGTGTCACTGTGCCTGCCCCTTGGATGAAGCGTGATGCATCTTCATGATG,, and the amino acid sequence of the protein product N207 of the PK5a1 hidden exon is shown as SEQ ID NO.2 ：MASDAVQSEPRSWSLLEQLGLAGADLAAPGVQQQLELERERLRREIRKELKLKEGAENLRRATTDLGRSLGPVELLLRGSSRRLDLLHQQLQELHAHVVLPDPAATHDGPQSPGAGGPTCSATNLSRVAGLEKQLAIELKVKQGAENMIQTYSNGSTKDRKLLLTAQQMLQDSKTKIDIIRMQLRRALQAGQLENQAAPDDTQGLAL*. Use of the PK5a1 harboring exon and protein product N207 marker described above for the preparation of a kit for detecting amyotrophic lateral sclerosis ALS. Preferably, the detection method of the kit for detecting amyotrophic lateral sclerosis ALS is qRT-PCR, RNA sequencing, immunoblotting, ELISA or mass spectrometry detection. The invention also discloses application of the PK5a1 hidden exon and protein product N207 marker in ALS and AD risk prediction systems. Preferably, the ALS and AD risk prediction system steps are: Firstly, extracting RNA or protein by a sample pretreatment module; secondly, detecting PK5a1 or N207 level by a molecular detection module; And thirdly, a data analysis module is used for establishing a prediction model by combining clinical indexes. Preferably, the clinical index is ALSFRS-R and/or neurofilament light chain NEFL level. Preferably, activation of PK5a1 in any of the above applications is dependent on the loss of TDP-43 function, and TDP-43 knockdown induces the production of the hidden exon of PK5a 1. The method has the advantages that 1, the high specificity is that PK5a1 activation depends on TDP-43 functional defects and hardly occurs in normal individuals, 2, the detectability is that N207 protein can be detected through ELISA, immunoblotting and mass spectrometry methods, the method is suitable for clinical transformation, in order to improve the detection specificity, N207 optimal antigen epitope is identified based on epitope prediction software Kolaskar-Tongaonkar, parker, karplus-Schulz and the like, as shown in SEQ ID NO.3, DDTQGLAL (a.a. 200-207), the peptide segment is completely absent in PKN1 full-length protein and can be used as an immune peptide for preparing monoclonal and polyclonal antibodies for detecting N207 and is used for WB, ELISA and body fluid detection, 3, the prediction value is that PK5a1/N207 and NEFL are combined, the early diagnosis accuracy of ALS and AD can be obviously improved, the method is suitable for preparing ALS and AD risk prediction systems, and 4, the PK5a1 hidden exon and protein product N207 marker are suitable for preparin