| Different signal transduction pathways in cells are regulated by a series of different proteomes,and upstream proteins regulate the activity of downstream proteins in each signaling pathway,a process that converts extracellular signals into intracellular signals.The cascade of signals is then amplified,dispersed,and conditioned to ultimately produce a comprehensive array of cellular responses,including activation or inhibition.Among numerous signal transduction pathways,autophagy,as a continuous homeostasis process,plays an important stabilizing role in cells and the whole organism.This process is capable of breaking down damaged cellular components,such as damaged organelles,misfolded proteins,and certain intracellular pathogens,which are depleted by autophagosomes to produce energy that can be supplied to the cells to maintain vitality and maintain homeostasis.Further research on autophagy-related molecules also links autophagy to human-related diseases.Many neurodegenerative diseases can be attributed to defects in autophagy function.For example,Parkinson's disease,amyotrophic lateral sclerosis,Alzheimer's disease,etc.,may be caused by abnormal autophagy function,resulting in the inability to clear aggregates of mutant toxic proteins.In addition,in autophagy and tumor related research,it is found that several proteins related to autophagy have tumor suppressor activity to some extent,but cancer cells may also provide survival mechanism for further development through autophagy pathway.In recent years,studies on the apoptotic pathway have found that apoptosis is a mechanism of programmed cell death.This process involves a signal cascade between various proteases,and is often accompanied by the production of different signaling molecules under different stimuli.These signaling molecules further regulate apoptosis,thereby clearing abnormal cells and achieving the goal of maintaining their overall homeostasis.In recent years,it has been found in the research on various diseases that if the death mechanism of diseased cells is further understood,the treatment scheme can be designed according to the death mechanism to effectively deal with the occurrence and development of diseases.Autophagy and apoptosis are the two main physiological pathways of cells,involving a large number of signal cascades and cross-regulation of signaling molecules.In studies of both pathways,autophagy is generally thought to promote cell survival,a process that protects cells from therapeutic drugs and allows cells to survive long-term starvation and other stimuli.However,there is a large of literature indicating that in some cases autophagy can kill cells by actively degrading essential cellular components(such as catalase,mitochondria)or by non-selective degradation of cellular components,so that cells no longer survive.This suggests that autophagy can promote death,which itself may be a mechanism of cell death.Therefore,it is of great significance to study the relationship between autophagy and apoptosis pathways at the molecular level,understand the whole cell process,and design targeted methods and drugs at the molecular level to achieve the treatment of diseases.In the study of these two processes,there are often some related markers,such as cathepsin B,caspase-3,ATG4B and so on.Through the study of these related proteins,we can further understand the mechanism of action of various proteins in the pathway.At present,the most commonly used researches on protein pathway related protein markers are biological means,such as Western Blot,PCR,etc.These research methods are cumbersome to operate,and often need to destroy the cell structure in the research,making it impossible to accurately detect the real-time changes of signal molecules in the pathway.In addition,some fluorescent probes have also been applied to the detection of signaling pathways,especially gold nanoprobes linked to thiol-modified DNA or polypeptide sequences by gold-sulfur bonds,and have been applied to the detection of signal molecules in various signal transduction pathways.However,in practical applications,the higher content of thiol in the organism can destroy the gold-sulfur bond,and cannot effectively visualize the true changes of the upstream and downstream signal molecules,thus affecting the authenticity and reliability of the signal molecule detection.Therefore,the development of a strong anti-interference fluorescent probe is of great significance for real-time in situ visual imaging detection of upstream and downstream markers in the process of autophagy-apoptosis.The gold-selenium bond has similar properties to the gold-sulfur bond,and found that selenol can break the gold-sulfur bond in the experiment,so as to form a more stable gold-selenium bond on the surface of gold.The problem of instability of the gold-sulfur bond in the organism can be solved by building a gold-selenium probe.Based on Au-Se bond,cathepsin B,caspase-3,ATG4B peptide chain,two gold nanoprobes capable of detecting autophagy-apoptosis related proteins were designed and synthesized.Specifically,it includes the following two aspects:1.Based on the Au-Se bond,we modified the peptide chain(labeled FITC)that specifically recognizes caspase-3 and the peptide chain(labeled 5-TAMRA)that specifically recognizes cathepsin B on Au NPs.After the probe is assembled,the fluorescence of the two fluorescent dyes is quenched by the gold nanoparticles.When the specific proteases cathepsin B and caspase-3 are incubated with the nanoprobe,the peptides can be specifically cleaved at the corresponding cleavage sites,thereby restoring the fluorescence previously quenched by the gold nanoparticles.Compared with gold-sulfur probes,gold-selenium probes have better resistance to GSH interference and high-temperature interference.More importantly,when the apoptotic pathway of breast cancer cells and liver cancer cells is activated by resveratrol,the fluorescence signal of the upstream cathepsin B and the fluorescence signal of the downstream caspase-3 can be observed by the Au-Se probe.It appeared in 4 hours,and the fluorescence intensity gradually increased with the increase of time.However,the Au-S probe could not observe the phenomenon of two upstream and downstream apoptosis markers due to the interference of reducing substances such as GSH.The cells treated with the same drug were then subjected to Western Blot experiments.The results were consistent with the results of confocal observations,which proved that Au-Se nanoprobes can be used to monitor the upstream and downstream relationships of the two signal molecules.In general,the design and preparation of this Au-Se nanoprobe provides a new strategy for real-time in situ study of the upstream and downstream relationship of signaling molecules in intracellular signal transduction pathways.2.Based on the Au-Se bond,we optimized the synthetic gold selenium probe based on the freezing method,we successfully modified the peptide chain(labeled FITC)that specifically recognizes caspase-3 and the peptide chain(labeled 5-TAMRA)that specifically recognizesATG4B on Au NPs,which not only greatly shortens the time of probe preparation,but also further increases the number of peptide chains attached to the probe.At this time,the fluorescence of the two fluorescent dyes can be quenched by the gold nanoparticles.When the specific proteases ATG4B and caspase-3 are encountered,the peptide chains can be specifically cleaved at the corresponding cleavage sites,thereby restoring the fluorescence previously quenched by the gold nanoparticles.The Au-Se probe has better anti-GSH interference ability,so that it can stably exist in the high bio-thiol environment of the cell,so that the expression of autophagy or apoptotic protein produced by the different drug treatment can be detected in real time,providing a new strategy for the detection of related proteins in cell signaling pathways in the future. |
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