Proteomics has the potential to provide answers in the pathogenesis of cancer and autoimmune disease by comprehensively analysing protein expression levels and activation statuses. In our study, we employed a quantitative proteomics strategy to compare the autophagy-related differentially expressed proteins of BMMCs in ITP patient and control groups and to explore the potential mechanism of ITP pathogenesis. The data showed that 26 upregulated proteins in ITP were enriched in the acute-phase response and regulation of the adaptive immune response, while 69 downregulated proteins were enriched in processes associated with binding, such as actin binding and cytoskeletal protein binding, which manifested as changes in the autophagy fractions. Then, we detected 5 abnormally expressed autophagy-related proteins that may be associated with the pathogenesis of ITP, among which 4 proteins (HSPA8, PARK7, YWHAH, and ITGB3) were downregulated and 1 protein (CSF1R) was significantly upregulated in ITP patients compared with those in controls. Clustering analysis showed that most of the autophagy-related differentially expressed proteins (YWHAH, ITGB3 and CSF1R) in this research were closely related to the PI3K/Akt/mTOR signalling pathway. The signalling pathway targeting mammalian target of rapamycin (mTOR) mediates many physiological functions, such as cell proliferation, differentiation, migration and apoptosis, and constitutes an important signalling pathway that regulates autophagy[18, 19]. Studies have shown that the pathway controlling mTOR expression negatively regulates autophagy in cells stimulated by factors such as starvation and hypoxia.
YWHAH is a large family of phosphoregulatory proteins that exist primarily as homo- and heterodimers. YWHAH proteins are involved in different signalling pathways that modulate cellular and whole-body energy and nutrient homeostasis, such as insulin signalling and mTOR- and AMP-dependent kinase signalling pathways (AMPK pathway), and regulate autophagy. There is considerable cross-talk between the AMPK pathway and other key energy regulatory pathways, such as insulin signalling and mTOR signalling complex 1 (mTORC1). AMPK is reported to inhibit mTORC1 by activating TSC1/2 (tuberous sclerosis protein 1 and 2) and by inhibiting regulatory-associated protein of TOR (RAPTOR) by phosphorylation-induced binding of YWHAH, both of which will stimulate autophagy. Recently, a direct stimulatory path from AMPK to autophagy was described through the phosphorylation of ULK1, and complex formation between ULK1, mTORC1 and AMPK has been found to coincide with the phosphorylation of RAPTOR and binding of YWHAH. We speculated that abnormal autophagy was associated in ITP patients with low expression of the YWHAH protein by the inhibition of RAPTOR, ultimately decreasing the function and quantity of megakaryocytes and platelets and leading to the onset of ITP.
KEGG enrichment analysis of differentially expressed proteins showed that the downregulated, autophagy-related protein ITGB3 was also enriched in platelet activation and haematopoietic cell lineage. ITGB3 is an important molecule involved in cell survival, proliferation and cancer metastasis. ITGB3 has been reported to be upstream of the PI3K/AKT/mTOR signalling pathway in various cell types, and the pathway is activated when ITGB3 is overexpressed. Studies have shown that ITGB3 upregulation inhibits the autophagic process in cardiomyocytes by activating AKT, suggesting that the expression status of ITGB3 may affect cell autophagy. Our results showed that the expression of ITGB3 in the ITP patient group was lower than that in the control group, suggesting that the overexpression of autophagy may be caused by the downregulation of AKT activation by ITGB3.
Another important autophagy-related protein in this research is CSF1R. Autophagy mediated by CSF-1/CSF1R plays a crucial role during the differentiation of human monocytes into macrophages[29, 30], which induce typical autophagic structures, such as phagophores and autophagosomes, and results in the accumulation of LC3-II. Tian et al. showed that the level of LC3-II in cells was lower in cells overexpressing CSF1R-Mut than in benign controls or CSF1R-WT cells when exposed to CSF-1 stimulation, indicating that the autophagy process might be disturbed by abnormal CSF-1/CSF1R signalling. At the molecular level, E5[N-(3-((4(benzofuran-2-yl) pyrimidin-2-yl) oxy)-4-methylphenyl)-4-((4-methylpiperazin-1-yl) methyl) benzamide] was able to downregulate the mTOR pathway and to activate the MAPK/ERK pathway[17, 32], thus inducing the conversion of LC3-I to LC3-II, increasing the expression of Atg5 and restoring autophagy. In our results, the expression level of CSF1R was decreased in the control group compared with that in the ITP patient group, which may demonstrate that the abnormal autophagy mediated by CSF1/CSF1R signalling is involved in the pathogenesis of ITP.
HSPA8 was enriched in the longevity regulating pathway - multiple species in KEGG analysis. HSPA8 is a molecular chaperone involved in a wide variety of cellular processes and is located in the nucleus, the cytosol, extracellular exosomes, and the cell membrane. HSPA8 is a detector of substrates that will be processed by chaperone-mediated autophagy (CMA). HSPA8 expression is altered in a number of immune disorders. For example, flow cytometry studies showed that the expression of HSPA8 is increased in B cells and T cells in the spleen of MRL/MpTn-gld/gld lupus-prone mice[35, 36]. HSPA8 is also involved in the molecular regulation of haematopoiesis. PARK7 is a multifunctional protein involved in various cellular activities. One of its principle functions is antioxidative defence and maintains mitochondrial homeostasis. The dysfunction of PARK7 leads to mitochondrial defects. Furthermore, CMA protects cells from mitochondrial toxin MPPC-induced changes in mitochondrial morphology and function and increases cell viability. Under PARK7-deficient conditions in ITP, these protective effects may be lost.
In recent years, experimental and clinical evidence has concluded that autophagy plays an important role in maintaining the stemness and microenvironment of haematopoietic stem cells. Perturbations of normal autophagy processes in ITP patients may be caused by the deletion of autophagy-related genes such as ATG7 and abnormal signalling due to the overexpression of mTOR. These changes are thought to affect markers of haematopoietic stem cells, such as CD41 and CD61, and the differentiation of megakaryocytes, ultimately decreasing the function and quantity of platelets and leading to the onset of ITP. Ouseph et al. demonstrated that the autophagy process is essential for the normal functioning of platelet activation and aggregation. In another study, they demonstrated that starvation induced substantial autophagy (above basal level), which was characterized by decreased platelet aggregation, reduced calcium mobilization and granule secretion, and decreased adhesion to immobilized fibrinogen, and eventually increased bleeding time.
In conclusion, autophagy-related differentially expressed proteins were found in ITP BMMC samples. GO, KEGG, protein domain enrichment and clustering analyses were performed to determine the correlation between the function and differential expression of proteins. PRM analysis further confirmed that the expression of autophagy-related proteins was significantly different in ITP patients. Furthermore, we indicate that the five autophagy-related differentially expressed proteins were closely related to mTOR signalling or similar pathways to regulate autophagy activity, which may provide useful information for ITP diagnosis and targeted treatment in the future.