CN-121991241-A - Fusion protein for regulating and controlling islet beta cell function based on optogenetic technology and application thereof

Abstract

The invention discloses a fusion protein for regulating and controlling islet beta cell functions based on an optogenetic technology and application thereof, and relates to the field of biotechnology. The fusion protein Opto LOVTRAP has high space-time resolution, can realize rapid and accurate control of single cells by adjusting blue light, has reversibility Opto LOVTRAP, can effectively inhibit Ca 2+ channels of islet beta cells under dark conditions, and can stop acting quickly after blue light stimulation, thereby achieving the purpose of reversible and accurate regulation and control of insulin secretion. Opto LOVTRAP acts on the concentration of Ca 2+ in cells in a targeting way, is a final trigger factor for the release of islet vesicles, is not influenced by the function of K ATP , and achieves the aim of breaking through the bottleneck of a clinical traditional treatment mode.

Inventors

• FU JUNFEN
• PENG DANDAN
• XU YINGKE
• DENG XIN
• WANG JINMIN

Assignees

• 浙江大学

Dates

Publication Date

20260508

Application Date

20241101

Claims (10)

1. 1. A fusion protein for regulating and controlling functions of islet beta cells based on a optogenetic technology, which is characterized by comprising a first fusion protein and a second fusion protein, wherein the first fusion protein comprises a membrane localization sequence for anchoring a photosensitive protein LOV2 domain on islet beta cell membranes and a photosensitive protein LOV2 domain which are sequentially connected from N end to C end, the second fusion protein comprises a binding protein ZDK2 for binding the photosensitive protein LOV2 domain, a calcium channel inhibitor protein and a fluorescent protein which are sequentially connected from C end to N end, and the calcium channel inhibitor protein is used for inhibiting opening of a calcium channel; in the dark state, the photoactive protein LOV2 domain in the first fusion protein binds to the binding protein ZDK2 in the second fusion protein, and the calcium channel inhibitor protein in the second fusion protein inhibits the opening of calcium channels; under light conditions, the photoactive protein LOV2 domain in the first fusion protein and the binding protein ZDK2 in the second fusion protein dissociate, and the calcium channel inhibitor protein in the second fusion protein dissociates to open calcium channels.
2. 2. The fusion protein of claim 1, wherein the membrane localization sequence is MGCIKSKRKDNLNDDE.
3. 3. The fusion protein of claim 1, wherein the calcium channel inhibitor protein is amino acids 1-266 of the Rem protein, The amino acid sequence of the 1 st to 266 th amino acid sequence of the Rem protein is shown as the 252 th to 517 th amino acid sequence of SEQ ID No. 2.
4. 4. The fusion protein of claim 1, wherein the fluorescent protein is a red fluorescent protein or a blue fluorescent protein.
5. 5. The fusion protein of claim 1, wherein the amino acid sequence of the first fusion protein is shown in SEQ ID No.1 and the amino acid sequence of the second fusion protein is shown in SEQ ID No. 2.
6. 6. A gene encoding the fusion protein according to any one of claims 1 to 5.
7. 7. The gene of claim 6, wherein the gene sequence encoded by the first fusion protein is shown in SEQ ID No.3, and the gene sequence encoded by the second fusion protein is shown in SEQ ID No. 4.
8. 8. The use of the fusion protein according to any one of claims 1-5 in the preparation of a medicament for treating a disease caused by dysregulated insulin secretion from pancreatic beta cells.
9. 9. The use according to claim 8, wherein the disorder caused by dysregulated insulin secretion from islet β cells is congenital hyperinsulinemia.
10. 10. Use according to claim 9, wherein, in use, the plasmid or adenovirus containing the fusion protein is transfected into a recipient tissue or cell, and the recipient tissue or cell is subjected to light stimulation.

Description

Fusion protein for regulating and controlling islet beta cell function based on optogenetic technology and application thereof Technical Field The invention relates to the technical field of biology, in particular to a fusion protein for regulating and controlling islet beta cell functions based on optogenetic technology and application thereof. Background Congenital hyperinsulinemia (Congenital hyperinsulinism, CHI) is mainly caused by mutation of genes related to insulin secretion of islet beta cells, and causes dysfunction of insulin secretion of islet beta cells, and is characterized in that the islet beta cells can still secrete excessive insulin when the organism suffers from hypoglycemia. CHI is a major cause of infant refractory hypoglycemia, and patients often suffer from epilepsy, coma, etc. during neonatal or infant periods. This can lead to irreversible brain damage, and may even cause death of the child patient if the patient is not cured in time. The persistent and intractable hypoglycemia of the neonate is not diagnosed in time, and the risk of permanent brain injury of the CHI infant is as high as 25% -50%. The disease has complex etiology and wide clinical manifestation spectrum, and causes extremely high risk of the patient to develop nerve development disorder. Therefore, it is important to clarify the pathogenesis of CHI and to realize early diagnosis and treatment of congenital hyperinsulinemia. The current treatment regimens for CHI fall into the two categories of surgical and pharmaceutical treatments. The intervention means are limited, and the following problems are mainly presented (1) Adenosine Triphosphate (ATP) sensitive potassium ion channel (K ATP) dependence. Diazoxide is the only first-line drug approved by the U.S. food and drug administration (Food and Drug Administration, FDA) for the treatment of CHI, whose primary target is K ATP, an opener for potassium ion channels. Diazoxide is therefore ineffective against infants with a loss of function of the K ATP channel, and is effective only against CHI caused by the K ATP mutation, which is either complete or dominant in the K ATP channel. However, most che is continuously generated mainly due to mutation of the K ATP channel gene, and the difference of the therapeutic effects of patients on diazoxide drugs is large due to the difference of key gene mutation sites. In addition, diazoxide has no pancreatic tissue specificity, which is prone to various adverse reactions. Such as fluid retention, pulmonary hypertension, etc., and serious cases may endanger the life of the infant. Its application is limited. (2) traditional medicine treatment is deficient. In addition to diazoxide, octreotide and other drugs are now also used for chronic treatment of children suffering from CHI, however, adverse reactions exist. Octreotide can cause abdominal discomfort, diarrhea, gall stones, cholecystitis and the like, can be used for reducing growth and relieving long QT syndrome of bradycardia, and can cause necrotizing colitis most seriously. Nifedipine has poor effect in clinical therapy of CHI and has obvious disadvantages including off-targeting, lack of spatial control and irreversibility as a Ca 2+ channel blocker. (3) postoperative complications are severe. If the medication fails, the child patient can usually only select the surgical treatment, which will bring about various postoperative complications. Focal che patients are cured by segmental or selective excision of the pancreas, but it should be noted that the majority of che patients receiving segmental pancreatectomy are at increased risk of developing insulin resistant diabetes. Diffuse chei often requires a near-holofilmectomy, and near two thirds of chei can have problems such as exocrine pancreatic insufficiency, poor postoperative efficacy, and more complications. Under physiological conditions, the K ATP channel is in an open state in resting islet beta cells, and potassium ions outflow, keeping the resting beta cells in a hyperpolarized state. When the glucose level in the plasma is increased, glucose taken up by beta cells is glycolyzed, and the intracellular ATP/ADP proportion is increased together with a plurality of biochemical processes in mitochondria, and the ATP is combined with Kir6.2 on a K ATP channel to promote the K ATP channel to be closed, so that the depolarization of islet beta cell membranes is triggered, a voltage-gating type Ca 2+ channel is triggered to be opened, the intracellular Ca 2+ concentration is increased, the exocytosis effect of intracellular Ca 2+ dependent insulin secretion particles is caused, the insulin release is promoted, and the abnormal process of any of the above processes can cause CHI to occur. The increased intracellular Ca 2+ concentration is the final trigger for insulin vesicle release and therefore, inhibition of voltage-gated Ca 2+ channels can be one of the effective ways to treat CHI. The optogenetics combines optics an